CN105050623B - The combination of PI3K inhibitor and C-MET inhibitor - Google Patents

Abstract

The present invention relates to include following pharmaceutical composition：(a) 3 kinase inhibitor of phosphatidylinositols or its pharmaceutically-acceptable salts, (b) at least one c Met receptor tyrosine kinase inhibitors or its pharmaceutically-acceptable salts, for simultaneously, separately or sequentially applying to treat proliferative diseases, particularly c Met dependences proliferative diseases；Include the pharmaceutical composition of the combination；The method of object of the treatment with proliferative diseases, the method includes applying the combination to object in need；Purposes of the combination in proliferative diseases are treated；And the commercial package comprising the combination.

Description

The combination of PI3K inhibitor and C-MET inhibitor

Technical field

The present invention relates to comprising following pharmaceutical composition, including (a) inhibitors of phosphatidylinositol3 3-kinase or its pharmacy Upper acceptable salt and (b) at least one c-Met receptor tyrosine kinase inhibitors or its pharmaceutically-acceptable salts, for simultaneously, It separates or sequence is applied to treat proliferative diseases, particularly c-Met dependences proliferative diseases；Include the drug of the combination Composition；The method of object of the treatment with proliferative diseases, including applying the combination to object in need；The combination is being controlled Treat the purposes in proliferative diseases；And the commercial package comprising the combination.

Background technology

Protein kinase (PK) is the enzyme of the diversified and important bioprocess of one group of adjusting, and the bioprocess includes thin Intracellular growth, survival and differentiation, orga- nogenesis and form generation, neovascularization, tissue repair and regeneration etc..Protein kinase passes through The phosphorylation of catalytic protein (or substrate) plays its physiological function so as to adjust the cell activity of substrate under different biological contexts.

Protein kinase can be divided into receptor type and non-receptor type.Receptor tyrosine kinase (RTK) has extracellular portion, cross-film knot Structure domain and intracellular part, and nonreceptor tyrosine kinase is entirely intracellular.RTK mediation signal transduction normally start from specifically Extracellular interaction occurs for growth factor (ligand), is usually followed by Receptor dimerization, stimulates integrated protein tyrosine-kinase enzyme activity Property and receptor turn phosphoric acid.So as to form binding site for intracellular signal transduction molecule and cause and a series of cytoplasmic signaling molecules Complex is formed, promotes suitable cell reaction such as cell division, differentiation, metabolic effect and extracellular micro-environmental variation.Non-receptor type Tyrosine kinase is also made of multiple subfamilies, including Src, Btk, Abl, Fak and Jak.

Immunohistochemistry is unique different dimerization receptor tyrosine kinase subfamily member, including Met, Ron and Sea (Birchmeier, C. etc., Nat.Rev.Mol.Cell Biol.2003,4 (12):915-925；Christensen, J.G. etc., Cancer Lett.2005,225(1):1-26).Unique high-affinity ligand of c-Met is hepatocyte growth factor (HGF), Referred to as dispersion factor (SF).HGF combinations c-Met can induce the receptor activation through autophosphorylation, cause receptor-independent signal Conduction increases.C-Met and HGF wide expressions are in a variety of organs, but its expression is usually limited to epithelium and mesenchyma source respectively Cell.The biological function of c-Met (or c-Met signal paths) in normal structure and human malignancies such as cancer has in detail It is thin to record (Christensen, J.G. etc., Cancer Lett.2005,225 (1):1-26；Corso, S. etc., Trends in Mol.Med.2005,11(6):284-292)。

Normal mammalian development needs HGF and c-Met, is reported in the mouse of HGF- missings and c-Met- missings different Often consistent with the nearly embryo expression in organ morphology generating process and Epithelial and stromal conversion defect (Christensen, J.G. etc., Cancer Lett.2005,225(1):1-26).It is consistent with these discoveries, the signal transduction of HGF/c-Met accesses and with Biological effect afterwards show to during development Epithelial and stromal interaction and to cell migration, invasion, cell Proliferation and Survival, angiogenesis, form occurs and the adjusting (such as renal tubular cell, gland is formed) of three-dimensional tube structure composition is important.c- Specific outcome of the Met signal pathway activateds in given cell/tissue is highly context dependence.

Evidence show the c-Met accesses of imbalance are formed in tumour, growth, maintain and progress in play an important role and sometimes It is the origin cause of formation (in the case of gene alteration) ((Birchmeier, C. etc., Nat.Rev.Mol.Cell.Biol.2003,4 (12): 915-925；Boccaccio, C. etc., Nat.Rev.Cancer 2006,6 (8):637-645；Christensen, J.G. etc., Cancer Lett.2005,225(1):1-26).Mistake in quite a few of HGF and/or c-Met in most of human cancers Expression, and usually related to bad clinical effectiveness, such as more serious disease, progression of disease, metastases and the patient of shortening survival Phase.In addition, the high patient of HGF/c-Met protein levels to chemotherapy and radiation more tolerant to.In addition to abnormal HGF/c-Met is expressed, c- Met receptors can also be activated by gene mutation (germ line mutation and somatic mutation) and gene magnification in cancer patient.Although Gene magnification and mutation are the most common gene alterations reported in patient, and the receptor can also be by lacking, blocking and gene weight Row and abnormal receptor processing are activated with deficiency negative regulation mechanism.

In addition, after activation, cMET activates a variety of intracellular signaling pathways, including but not limited to phosphatidyl-inositol 3-kinase (PI3K)/Akt accesses, ERK 1/2, p38 and STAT3.PI3K includes lipid and serine/threonine kinase family, catalysis Phosphoric acid is transferred to the D-3 ' positions of inositol lipid to generate phosphatidylinositols -3- phosphoric acid (PIP), phosphatidylinositols -3,4- diphosphonic acid (PIP2) and phosphatidylinositols -3,4,5- triphosphoric acids (PIP3), so by make domain containing pleckstrin homology, FYVE, Phox and the albumen of other phosphatide binding domain are stopped into serving as signal cascade in a variety of signal complexs for being usually located at plasma membrane The second messenger (, Annu.Rev.Biochem such as Vanhaesebroeck 70:535(2001)；The such as Katso, Annu.Rev.Cell Dev.Biol.17:615(2001)).In two kind of 1 type PI3K, 1A types PI3K be form as follows it is heterodimeric Body：Catalysis p110 subunits (α, β, δ isomers) with adjust subunit composing type be connected, it is described adjust subunit can be p85 α, p55 α, P50 α, p85 β or p55 γ.There are one family members for 1B types subclass tool, i.e., adjust subunit p101 by being catalyzed p110 γ subunits and 2 Or one of p84 is connected the heterodimer (, Annu such as Fruman Rev.Biochem.67 of composition:481(1998)；The such as Suire, Curr.Biol.15:566(2005)).The modular structural domains of p85/55/50 subunits include homologous (SH2) structural domains of Src, the knot Structure domain combines the phosphotyrosine residue on activated receptor and cytoplasmic tyrosine kinase under particular sequence background, causes 1A types The activation and positioning of PI3K.1B types PI3K by g protein coupled receptor direct activation, the receptor combine a diversified peptide and Non-peptide ligand library (the, Cell such as Stephens 89:105(1997))；The such as Katso, Annu.Rev.Cell Dev.Biol.17: 615-675(2001)).PI3K accesses are the important regulatory factors of critical cellular processes, and the cell processes include proliferation, deposit Work, chemotaxis, cell transport, motility, metabolism, inflammatory and allergic reaction, the transcription and translation (, Cell such as Cantley 64: 281(1991)；Escobedo and Williams, Nature 335:85(1988)；The, Cell., 69 such as Fantl:413 (1992).)。

Although there are remarkable break-throughs in terms of the therapeutic choice of medicine and many cancer patients, effect and safety are needed remain for Therapeutic agent and the new combination treatment that effective long-term treatment cancer can be applied to.It has now found that comprising following combination to treatment Proliferative diseases, particularly c-Met dependences proliferative diseases are particularly effective：(a) selected from formula (I) compound, formula (II) chemical combination The inhibitors of phosphatidylinositol3 3-kinase or its pharmaceutically-acceptable salts of object and formula (III) compound and (b) at least one c- The fluoro- N- methyl -4- of Met receptor tyrosine kinase inhibitors, particularly 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] -2 base of triazine] benzamide or its pharmaceutically-acceptable salts.It is expected that the anti-proliferative effect of this combination is higher than any sort list Alone become a point attainable maximum efficiency.

Invention content

The present invention is about a kind of pharmaceutical composition, including (a) selected from formula (I) compound, formula (II) compound and formula (III) The phosphatidyl-inositol 3-kinase of compound or the suppression of its pharmaceutically-acceptable salts and (b) at least one c-Met receptor tyrosine kinases Preparation or its pharmaceutically-acceptable salts, for simultaneously, separate or sequence application with treat proliferative diseases, particularly c-Met according to Rely property proliferative diseases.Present invention combination is particularly useful for the treatment of lung cancer (such as non-small cell lung cancer) or spongioblastoma.

In a preferred embodiment, formula (I) compound is 2- methyl -2- [4- (3- methyl -2- oxygen -8- quinoline -3- Base -2,3- dihydro-imidazols simultaneously [4,5-c] quinoline -1- bases)-phenyl]-propionitrile (" compound A ") or its monotosylate salt and 8- (6- methoxv-pyridine -3- bases) -3- methyl-1s-(4- piperazine -1- base -3- trifluoromethyl-phenyls) -1,3- dihydro-imidazols are simultaneously [4,5-c] quinoline-2-one (" compound B ").

In a preferred embodiment, formula (II) compound is 5- (2,6- bis--morpholine -4- bases-pyrimidine-4-yl) -4- Trifluoromethylpyridin -2- bases amine (" compound C ") or its hydrochloride.

In a preferred embodiment, formula (III) compound is (S)-pyrrolidines -1,2- dicarboxylic acids 2- amide 1- ({ 4- Methyl -5- [2- (tri- fluoro- 1,1- dimethyl-ethyIs of 2,2,2-)-pyridin-4-yl]-thiazol-2-yl }-amide) (" compound D ") Or its pharmaceutically-acceptable salts.

In a preferred embodiment, c-Met receptor tyrosine kinase inhibitors are fluoro- N- methyl -4- [the 7- quinolines of 2- Quinoline -6- bases-methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide or its pharmaceutically-acceptable salts.

On the one hand, the present invention relates to the methods for the treatment of proliferative diseases, and the method includes being applied to object in need The following combination of the amount of the proliferative diseases is effectively resisted in treatment：(a) selected from formula (I) compound, formula (II) compound and The phosphatidyl-inositol 3-kinase or its pharmaceutically-acceptable salts of formula (III) compound and (b) at least one c-Met receptor junket ammonia Acid kinase inhibitor or its pharmaceutically-acceptable salts.Preferably, the proliferative diseases are c-Met dependence proliferative diseases.

In another embodiment, the present invention relates to inhibit to suffer from the method for shifting formation in cancer object, the method packet Include the following combination for the amount that the proliferative diseases are effectively resisted in object in need application treatment：(a) change selected from formula (I) Conjunction object, the phosphatidyl-inositol 3-kinase of formula (II) compound and formula (III) compound or its pharmaceutically-acceptable salts and (b) are extremely A kind of few c-Met receptor tyrosine kinase inhibitors or its pharmaceutically-acceptable salts.Preferably, the cancer is that c-Met is relied on Property cancer.

On the one hand, proliferative diseases are treated the present invention relates to present invention combination in treatment proliferative diseases and/or preparing Purposes in drug.In a preferred embodiment, the proliferative diseases are cancers.In a preferred embodiment, The proliferative diseases are c-Met dependence proliferative diseases.

On the one hand, it is combined the present invention relates to the present invention and suffers from transfer formation in cancer object in inhibition and/or preparing inhibition trouble The purposes in the drug of formation is shifted in cancer object.In a preferred embodiment, the cancer is c-Met dependence cancers Disease.

On the one hand, the present invention provides the drug containing quantitative present invention combination and one or more pharmaceutically acceptable carriers Composition, the amount effectively resist proliferative diseases, particularly c-Met dependences proliferative diseases jointly in the treatment.This medicine Compositions can be made of therapeutic agent (a) and (b), be passed through as fixed Combination with single formulation or unit dosage forms any appropriate Approach be applied to object.Alternatively, the therapeutic agent such as inhibitors of phosphatidylinositol3 3-kinase and c-Met receptor tyrosine kinases Inhibitor or its pharmaceutically-acceptable salts are applied as non-fixed combinations with separated pharmaceutical composition or preparation or unit dosage forms For object, and without specific time limitation ground simultaneously, synchronous or sequence applies.

On the one hand, the present invention provides a kind of commercial package, and it includes the present invention combination as therapeutic agent and explanations Book, the specification are used for same in treatment proliferative diseases particularly lung cancer (such as non-small cell lung cancer) or spongioblastoma When, separate or sequence application the present invention combination.

Description of the drawings

The fluoro- N- methyl -4- of Fig. 1 display compound C and 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] - 2 base of triazine] benzamide unitized dose to the effect that is proliferated in H4 human glioblastoma's models.

The fluoro- N- methyl -4- of Fig. 2 display compound C and 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] - 2 base of triazine] benzamide unitized dose to the effect that is proliferated in U87-MG human glioblastoma's models.

The fluoro- N- methyl -4- of Fig. 3 display compound C and 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] - 2 base of triazine] benzamide unitized dose to the effect that is proliferated in A172 human glioblastoma's models.

The fluoro- N- methyl -4- of Fig. 4 display compound C and 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] - 2 base of triazine] benzamide unitized dose to the effect that is proliferated in LN229 human glioblastoma's models.

Fig. 5 shown compared with supporting agent control and two kinds of single therapies, the fluoro- N- methyl -4- of 2- [7- quinoline -6- bases-methyl) - - 2 base of imidazo [1,2-b] [1,2,4] triazine] benzamide and compound D combination in NCI-H1993 human non-small cell's lungs Comparison in cancer heteroplastic transplantation model is averaged neoplasm growth effect.

Detailed description of the invention

The present invention relates to a kind of pharmaceutical composition, including：(a) selected from formula (I) compound, formula (II) compound and formula (III) phosphatidyl-inositol 3-kinase of compound or its pharmaceutically-acceptable salts and (b) at least one c-Met receptor tyrosines Kinase inhibitor or its pharmaceutically-acceptable salts, for simultaneously, separately or sequentially applying to treat proliferative diseases, particularly c- Met dependence proliferative diseases.Present invention combination is especially suitable for treatment lung cancer (such as non-small cell lung cancer) or spongioblast Knurl.

Unless expressly stated otherwise, general terms used herein are defined with following meanings：

Unless otherwise indicated, term "comprising" and " comprising " are used with open and unlimited meaning herein.

Term " one " and " described " and class in the context (the especially context of claim) of the description present invention It seemingly refers to and is regarded as covering odd number and plural number, in addition to this otherwise noted or is apparently contradicted in the context.Plural form is used for chemical combination Whens object, salt etc., it is also represented by single compound, salt etc..

Term " combination " or " pharmaceutical composition " are defined as in the single dose unit form for administering drug combinations herein Fixed Combination, non-fixed combinations or the medicine box containing each section, wherein inhibitors of phosphatidylinositol3 3-kinase and c-Met receptor junket Histidine kinase inhibitor or its pharmaceutically-acceptable salts can be applied simultaneously separately or therapeutic agent allowed to show cooperation (as assisted It is administered alone in the time interval of effect together).

Term " fixed Combination " refers to therapeutic agent, such as inhibitors of phosphatidylinositol3 3-kinase and c-Met receptor tyrosine kinases Enzyme inhibitor is administered simultaneously with single entity or dosage form in object.

Term " non-fixed combinations " refers to the therapeutic agent, such as inhibitors of phosphatidylinositol3 3-kinase and c-Met receptor junket Histidine kinase inhibitor or its pharmaceutically-acceptable salts, as corpus separatum or dosage form without specific time limitation ground simultaneously, it is synchronous Or sequence is applied to object, wherein described be applied in object in need such as mammal or human body provides treatment effective level 3 kinds of compounds.

Term " medicine box containing each section " refers to therapeutic agent (a) defined above and (b), individually or by using with not The different fixed Combinations of same amount therapeutic agent (a) and (b) prepare dosage, i.e., simultaneously or in different time points.Then, medicine box Each section energy, such as intersect application simultaneously or in chronological order, i.e., have in different time points and for any medicine box portion point Identical or different time interval.Therapeutic agent (a) to be administered and the variable-scale of therapeutic agent (b) total amount in combination preparation, Such as in order to meet the demand of the needs of patient subgroups to be treated or single patient.

Term " inhibitors of phosphatidylinositol3 3-kinase " or " PI3K inhibitor " are defined herein as targeting, reduce or pressing down The compound of phosphatidyl-inositol 3-kinase processed.Phosphatidyl-inositol 3-kinase activity has shown many hormones of response and growth factor It stimulates and increases, including insulin, platelet derived growth factor, insulin-like growth factor, epidermal growth factor, colony thorn Swash the factor and hepatocyte growth factor, and participate in the process for being related to cell growth and conversion.

Term " c-Met receptor tyrosine kinase inhibitors " is defined herein as targeting, reduce or inhibiting c-Met receptors The compound of tyrosine kinase activity.

Term " c-Met dependences proliferative diseases " or " c-Met dependence cancers " refer to c-Met and/or HGF/c-Met letters Those proliferative diseases of number access imbalance, particularly HGF/c-Met gene overexpressions and amplification, HGF- dependences autocrine and Paracrine activates and/or c-Met receptor mutations (germ line mutation and somatic mutation).In a preferred embodiment, The proliferative diseases are the cancers of c-Met and/or HGF/c-Met signal paths imbalance." c-Met and/or HGF/c-Met letters Number access imbalance " is intended to include through number of mechanisms activation c-Met receptoroid tyrosine kinase, and the mechanism includes but not limited to HGF- dependences autocrine and paracrine activation, point mutation, is lacked, is blocked, again HGF/c-met gene overexpressions and/or amplification Row and the processing of exception c-Met receptors and deficiency negative regulation mechanism.In a preferred embodiment, " c-Met is relied on term Property proliferative diseases " or " c-Met dependence cancers " are including HGF/c-met gene overexpressions and amplification.

Term " pharmaceutical composition " is defined herein as containing at least one to be administered in object such as mammal or people Therapeutic agent mixture or solution, for treat influence object specified disease or illness.

Term " pharmaceutically acceptable " is defined herein as being adapted for contact with object in scope of sound medical judgment as fed Newborn animal or those compounds, material, composition and/or the dosage form of people's tissue, without excessive toxicity, stimulation, allergic reaction With other problems complication, and with rational benefit/risk ratio.

The term as used herein " treatment " or " processing " are including alleviating, reducing or improving at least one object symptom or cause The treatment of progression of disease delay.For example, treatment can be reduced one or more disease symptoms or completely eliminate disease, such as cancer Disease.In the range of meaning of the present invention, term " treatment " also refers to retardance, delay disease occurs (stage i.e. before Disease Clinical performance) And/or reduce disease development or the risk deteriorated.

The term as used herein " co-therapies activity " or " combination therapy effect " refer to therapeutic agent can be between certain time Every separating (using interleaved mode, particularly sequence specificity pattern in chronological order) application, so as to which it is in homoiothermy to be treated Interaction (combination therapy effect) is preferably still shown and (preferably cooperateed with) in animal especially people.Whether situation so can be under Row blood level measures, and 2 kinds of therapeutic agents of display are collectively resided at least some of time interval in the blood of people to be treated.

Term " pharmaceutical effective amount " or the therapeutic agent drug of " clinical effective " combination refer to, compared to the combination institute Treat baseline clinical observable sign and the symptom of proliferative diseases, it is sufficient to provide the amount being obviously improved.

The term as used herein " synergistic effect " refers to the effect that 2 kinds of therapeutic agents generate a certain effect, and the effect for example subtracts The symptom progress of slow proliferative diseases especially cancer or its symptom are more than the simple folded of each therapeutic agent effect of application alone Add, 2 kinds of therapeutic agents such as (a) formula (I) compound such as compound A or compound B or its pharmaceutically-acceptable salts and at least A kind of c-Met receptor tyrosine kinase inhibitors or its pharmaceutically-acceptable salts or (b) formula (II) compound such as compound C Or its pharmaceutically-acceptable salts and c-Met receptor tyrosine kinase inhibitors or its pharmaceutically-acceptable salts or (c) formula (III) Compound, compound D or its pharmaceutically-acceptable salts and c-Met receptor tyrosine kinase inhibitors or its is pharmaceutically acceptable Salt.Synergistic effect can with suitable method such as Sigmoid-Emax equations (Holford, N.H.G. and Scheiner, L.B., Clin.Pharmacokinet.6:429-453 (1981)), Loewe sum it up equation (Loewe, S. and Muischnek, H., Arch.Exp.Pathol Pharmacol.114:313-326 (1926)) and middle effect formula (Chou, T.C. and Talalay, P., Adv.Enzyme Regul.22:27-55 (1984)) it calculates.Above-mentioned each equation can apply to experimental data to generate correspondence graph So as to assist the effect of evaluation pharmaceutical composition.Correspondence graph with above-mentioned dependence among equations is concentration-effect curve, equivalent line respectively Figure curve and combinatorial index curve.

The term as used herein " object " or " patient " include animal, can suffer from proliferative diseases or it is any directly or It connects the disease for being related to tumour or is affected by it.The example of object includes mammal, such as people, dog, ox, horse, pig, sheep, mountain Sheep, cat, mouse, rabbit, rat and transgenic nonhuman animal.In a preferred embodiment, the object is people, such as suffer from, The risky people suffered from or can potentially suffer from proliferative diseases.

Term " about " or " substantially " should be represented in the 10% of given value or range, more preferably in given value or range In 5%.

The combination of the present invention includes the PI3K selected from formula (I) compound, formula (II) compound and formula (III) compound and inhibits Agent or its pharmaceutically-acceptable salts.

WO2006/122806 and WO2008/103636 describe imidazoquinoline derivative, it is found that it inhibits PI3K and lactation to move The activity of object rapamycin target protein (mTOR).Be suitble to the present invention specific imidazoquinoline derivative, its prepare and containing its Suitable pharmaceutical formulation is described in WO2006/122806 and WO2008/103636, and including formula (I) compound

Wherein

R₁It is naphthalene or phenyl, wherein the phenyl is independently selected from 1 of halogen or 2 substituent groups replace；It is not taken Generation or the low alkyl group replaced by halogen, cyano, imidazole radicals or triazolyl；Cycloalkyl；It is independently selected from 1 or 2 of the following group The amino of substituent group substitution：Low alkyl group, lower alkylsulfonyl radicals, lower alkoxy and lower alkoxy loweralkyl amino； The piperazinyl that unsubstituted or being independently selected from low alkyl group and lower alkylsulfonyl radicals 1 or 2 substituent groups replace；2- oxygen Generation-pyrrolidinyl；Lower alkoxy loweralkyl；Imidazole radicals；Pyrazolyl；And triazolyl；

R₂It is O or S；

R₃It is low alkyl group；

R₄It is unsubstituted or is not taken by pyridyl group that halogen, cyano, low alkyl group, lower alkoxy replace or Generation or the piperazinyl replaced by low alkyl group；Pyrimidine radicals that is unsubstituted or being replaced by lower alkoxy；It is unsubstituted or by halogen The quinolyl of element substitution；Quinoxalinyl；Or the phenyl replaced with alkoxy；

R₅It is hydrogen or halogen；

N is 0 or 1；

R₆It is oxidation base；

If additional conditions are n=1, group R is carried₆N atoms have positive charge；

R₇It is hydrogen or amino.

Formula (I) compound defines group used and symbol with the meaning as disclosed in WO2006/122806, passes through Incorporated is incorporated herein.

A kind of PI3K inhibitor, that is, formula (I) compound can be present in group in the form of free alkali or its pharmaceutically-acceptable salts In conjunction.The acceptable acid addition salts of formula (I) compound include for example as acid-addition salts formed those, it is preferable to use organic or inorganic acid. For example, suitable inorganic acid is halogen acids, such as hydrochloric acid, sulfuric acid or phosphoric acid.For example, suitable organic acid is carboxylic acid, phosphonic acids, sulfonic acid Or sulfamic acid, as acetic acid, propionic acid, octanoic acid, capric acid, dodecanoic acid, glycolic, lactic acid, fumaric acid, succinic acid, malonic acid, oneself Diacid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, citric acid, amino acid such as glutamic acid or aspartic acid, Malaysia Acid, hydroxymaleic acid, citraconic acid, cyclohexane-carboxylic acid, adamantanecarboxylic acid, benzoic acid, salicylic acid, 4-ASA, neighbour Phthalic acid, phenylacetic acid, mandelic acid, cinnamic acid, methanesulfonic acid or ethanesulfonic acid, 2- ethylenehydrinsulfonic acids, ethane -1,2- disulfonic acid, Benzene sulfonic acid, 4- toluenesulfonic acids, 2- naphthalene sulfonic acids, 1,5- naphthalenedisulfonic acids, 2- or 3- toluenesulfonic acids, methylsulfuric acid, ethyl sulfuric acid, 12 Alkylsurfuric acid, N- cyclohexylsulfamics, N- methyl-, N- ethyls or N- propyl-aminos sulfonic acid or other organic proton acid such as Ascorbic acid.

Preferred formula (I) compound combined for the present invention is 2- methyl -2- [4- (3- methyl -2- oxygen -8- quinoline -3- Base -2,3- dihydro-imidazols simultaneously [4,5-c] quinoline -1- bases)-phenyl]-propionitrile (" compound A ") or its monotosylate salt and 8- (6- methoxv-pyridine -3- bases) -3- methyl-1s-(4- piperazine -1- base -3- trifluoromethyl-phenyls) -1,3- dihydro-imidazols are simultaneously [4,5-c] quinoline-2-one (" compound B ").For example, the synthesis of compound A and its monotosylate salt are described in In the embodiment 7 and 152-3 of WO2006/122806.For example, the synthesis of compound B is described in the embodiment of WO2006/122806 In 86.In a preferred embodiment, formula (I) compound is compound A or its monotosylate salt.

WO07/084786 descriptions are found that the pyrimidine derivatives of PI3K activity can be inhibited.The suitable present invention combines specific PI3K inhibitor, its prepare and be described in WO07/084786, and including formula (II) compound containing its suitable pharmaceutical formulation：

Wherein W is CR_wOr N, wherein

R_wIt is selected from the group：

(1) hydrogen,

(2) cyano,

(3) halogen,

(4) methyl,

(5) trifluoromethyl,

(6) sulfonamide；

R₁It is selected from the group：

(1) hydrogen,

(2) cyano,

(3) nitro,

(4) halogen,

(5) substitution and unsubstituted alkyl,

(6) substitution and unsubstituted alkenyl,

(7) substitution and unsubstituted alkynyl,

(8) substitution and unsubstituted aryl,

(9) substitution and unsubstituted heteroaryl,

(10) substitution and unsubstituted heterocycle,

(11) substitution and unsubstituted cycloalkyl,

(12)-COR_1a,

(13)-CO₂R_1a,

(14)-CONR_1aR_1b,

(15)-NR_1aR_1b,

(16)-NR_1aCOR_1b,

(17)-NR_1aSO₂R_1b,

(18)-OCOR_1a,

(19)-OR_1a,

(20)-SR_1a,

(21)-SOR_1a

(23)-SO₂NR_laR_1b, wherein

R_1aAnd R_1bIt is independently selected from the following group：

(a) hydrogen,

(b) substitution or unsubstituted alkyl,

(c) substitution and unsubstituted aryl,

(d) substitution and unsubstituted heteroaryl,

(e) substitution and unsubstituted heterocycle and

(f) substitution and unsubstituted cycloalkyl；

R₂It is selected from the group：

(1) hydrogen,

(2) cyano,

(3) nitro,

(4) halogen,

(5) hydroxyl,

(6) amino,

(7) substitution and unsubstituted alkyl,

(8)-COR_2aAnd

(9)-NR_2aCOR_2b, wherein

R_2aAnd R_2bIt is independently selected from the following group：

(a) hydrogen and

(b) substitution or unsubstituted alkyl；

R₃It is selected from the group：

(1) hydrogen,

(2) cyano,

(3) nitro,

(4) halogen,

(5) substitution and unsubstituted alkyl,

(6) substitution and unsubstituted alkenyl,

(7) substitution and unsubstituted alkynyl,

(8) substitution and unsubstituted aryl,

(9) substitution and unsubstituted heteroaryl,

(10) substitution and unsubstituted heterocycle,

(11) substitution and unsubstituted cycloalkyl,

(12)-COR_3a,

(14)-NR_3aR_3b,

(13)-NR_3aCOR_3b,

(15)-NR_3aSO₂R_3b,

(16)-OR_3a,

(17)-SR_3a,

(18)-SOR_3a,

(19)-SO₂R_3a, wherein

R_3aAnd R_3bIt is independently selected from the following group：

(a) hydrogen,

(b) substitution or unsubstituted alkyl,

(c) substitution and unsubstituted aryl,

(d) substitution and unsubstituted heteroaryl,

(e) substitution and unsubstituted heterocycle and

(f) substitution and unsubstituted cycloalkyl；With

R₄It is selected from the group：

(1) hydrogen and

(2) halogen.

Formula (II) compound defines group used and symbol with the meaning as disclosed in WO07/084786, passes through Incorporated is incorporated herein.

PI3K inhibitor, that is, formula (II) compound can be present in combination in the form of free alkali or its pharmaceutically-acceptable salts In.The acceptable acid addition salts of formula (II) compound include but not limited to following：Acetate, adipate, alginates, citrate, asparagus fern Propylhomoserin salt, benzoate, benzene sulfonate, disulfate, butyrate, camphor hydrochlorate, camsilate, digluconate, ring Pentane propionate, lauryl sulfate, esilate, glucoheptose salt, glycerophosphate, Hemisulphate, enanthate, caproic acid Salt, fumarate, hydrochloride, hydrobromate, hydriodate, 2- isethionates, lactate, maleate, mesylate, Nicotinate, 2- naphthalene sulfonates, oxalates, embonate, pectate, persulfate, 3- phenylpropionic acids salt, picrate, Pivalate, propionate, succinate, sulfate, tartrate, rhodanate, tosilate and caprate.Equally, The Basic nitrogen-containing groups can use such as following reagent quaternized：Alkyl halide such as methyl-, ethyl-, propyl-and butyl chlorination Object, bromide and iodide；Dialkyl sulfate such as dimethyl-, diethyl-, dibutyl-and diamyl sulfate, it is long Chain halide such as decyl-, dodecyl-, myristyl-and stearyl chlorides, bromide and iodide, aralkyl Halide such as benzyl and phenethyl bromide, etc..

The acceptable acid addition salts of formula (II) compound include but not limited to the cation based on alkali and alkaline earth metal ions, such as sodium Salt, lithium salts, sylvite, calcium salt, magnesium salts, aluminium salt etc. and nontoxic ammonium, quaternary ammonium and amine cation, including but not limited to ammonium, tetramethyl Ammonium, etamon, methylamine, dimethylamine, trimethylamine, triethylamine, ethamine etc..It is used to form other representative organic amines of base addition salts Including diethylamine, ethylenediamine, ethanol amine, diethanol amine, piperazine, pyridine, picoline, triethanolamine etc. and basic amino acid example Such as arginine, lysine and ornithine.

Preferred formula (II) compound combined for the present invention is PI3K inhibitor 5- (2,6- bis--morpholine -4- bases-phonetic Pyridine -4- bases) -4- trifluoromethylpyridin -2- bases amine (" compound C " hereafter) or its hydrochloride.The synthesis description of compound C In the embodiment 10 of WO 2007/084786, content is incorporated herein by reference.

WO2010/029082 describes specific 2- formamide ring semicarbazide derivatives, is found to PI3K α-isotype With inhibitory activity.Specific 2- formamide ring semicarbazide derivatives, its preparation and the conjunction containing it that the suitable present invention combines Suitable preparation is described in WO2010/029082, and including formula (III) compound：

Wherein

A represents heteroaryl selected from the group below：

R¹Represent one of substituents：(1) it is unsubstituted or substitution, the C preferably replaced₁-C₇Alkyl, wherein described Substituent group is independently selected from one or more, preferably 1-9 following parts：Deuterium, fluorine or 1-2 following part C₃-C₅Cycloalkanes Base；(2) C optionally replaced₃-C₅Cycloalkyl, wherein the substituent group is independently selected from one or more, preferably 1-4 following portions Point：Deuterium, C₁-C₄Alkyl (preferably methyl), fluorine, cyano, amino carbonyl；(3) phenyl optionally replaced, wherein the substituent group is only It is vertical to be selected from one or more, preferably 1-2 following parts：Deuterium, halogen, cyano, C₁-C₇Alkyl, C₁-C₇Alkyl amino, two (C₁-C₇Alkyl) amino, C₁-C₇Alkyl amino-carbonyl, two (C₁-C₇Alkyl) amino carbonyl, C₁-C₇Alkoxy；(4) optionally Monosubstituted or bis substituted amine；Wherein described substituent group is independently selected from following part：Deuterium, C₁-C₇Alkyl is (unsubstituted or by one It is a or it is multiple selected from deuterium, fluorine, chlorine, hydroxyl substituent groups substitution), benzenesulfonyl it is (unsubstituted or (excellent by following one or more Select one) substitution：C₁-C₇Alkyl, C₁-C₇Alkoxy, two (C₁-C₇Alkyl) amino-C₁-C₇Alkoxy)；(5) sulphur of substitution Acyl group；Wherein described substituent group is selected from following part：C₁-C₇Alkyl is (unsubstituted or taken by one or more selected from deuterium, fluorine Replace for base), pyrrolidinyl it is (unsubstituted or replaced by one or more substituent groups selected from deuterium, hydroxyl, oxygroup；Particularly One oxygroup)；(6) fluorine, chlorine；

R²Represent hydrogen；

R³Represent (1) hydrogen, (2) fluorine, chlorine, the methyl that (3) optionally replace, wherein the substituent group is independently selected from one or more It is a, preferably 1-3 following parts：Deuterium, fluorine, chlorine, dimethylamino.

Formula (III) compound defines group used and symbol with the meaning as disclosed in WO2010/029082, leads to Incorporated is crossed to be incorporated herein.

Preferred compounds of the invention is the compound of the specific descriptions of WO2010/029082.The highly preferred chemical combination of the present invention Object be (S)-pyrrolidines -1,2- dicarboxylic acids 2- amides 1- (4- methyl -5- [2- (tri- fluoro- 1,1- dimethyl-ethyIs of 2,2,2-) - Pyridin-4-yl]-thiazol-2-yl }-amide) (herein referred as " compound D ") or its pharmaceutically-acceptable salts.The conjunction of compound D Into being described in the embodiment 15 of WO2010/029082.

Formula (III) compound can be used in the form of free alkali or its pharmaceutically-acceptable salts.Acceptable acid addition salts include for example from There are those formed in formula (III) compound of basic nitrogen atom as acid-addition salts, it is preferable to use organic or inorganic acid.Example Such as, suitable inorganic acid is halogen acids, such as hydrochloric acid, sulfuric acid or phosphoric acid.For example, suitable organic acid is carboxylic acid or sulfonic acid, it is such as rich Horse acid or methanesulfonic acid.

The combination of the present invention further includes at least one c-Met receptor tyrosine kinase inhibitors or its is pharmaceutically acceptable Salt.The example of suitable c-Met receptor tyrosine kinase inhibitors includes but not limited to the fluoro- N- methyl -4- of 2- [7- quinoline -6- Base-methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide, ARQ197 (tavantinib), AMG458, GSK1363089 (XL880 or foretinib), PF2341066 (gram azoles replaces Buddhist nun) or its pharmaceutically-acceptable salts.

Preferably, the c-Met receptor tyrosine kinase inhibitors combined for the present invention are formula (IV) compounds

Or its pharmaceutically-acceptable salts.Formula (IV) compound has the fluoro- N- methyl -4- of chemical name 2- [7- quinoline -6- Base-methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide.This compound and its synthetic method are disclosed in the world In the embodiment 7 of PCT Patent Application WO2008/064157, the content of the patent is incorporated herein by reference of text.

Formula (IV) compound can be used in the form of free alkali or its pharmaceutically-acceptable salts.Fluoro- N- methyl -4- [the 7- quinolines of 2- Quinoline -6- bases-methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] acceptable acid addition salts of benzamide include alkaline residue such as amine Mineral or acylate；Alkali or organic salt of acidic residues such as carboxylic acid etc..The pharmaceutically-acceptable salts of the present invention include common The parent compound for example formed from nontoxic inorganic or organic acid nontoxic salts.Preferably, formula (IV) compound is PCT special Salt form disclosed in profit application WO2009/143211, is incorporated herein by reference of text.The preferred salt shape of formula (IV) compound Formula includes the fluoro- N- methyl -4- of 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine -2 base] benzamide Dihydrochloride form and diphenyl sulfonic acid salt form.

Suitable c-Met receptor tyrosine kinase inhibitors further include：

(i.) ARQ197 (tavantinib) is (by Sankyo Co. (Daiichi Sankyo) and A Kuli (ArQule) develop), there is chemical formula：

(ii.) AMG458 (being developed by pacifying into (Amgen)), has chemical formula：

(iii.) GSK1363089 (also referred to as XL880 or foretinib) is (by GlaxoSmithKline PLC (GlaxoSmithKline) develop), there is chemical formula：

(iv.) PF2341066 (also referred to as gram azoles replaces Buddhist nun) (being developed by Pfizer (Pfizer)), has chemical formula：

The therapeutic agent structure determined by number, common name or trade name can be from standard outline《Merck index》("The Merck Index ") current edition or database such as international monopoly (Patents International) (such as the IMS worlds go out Version object (IMS World Publications)) it obtains.Its corresponding content is incorporated herein by reference.

Present invention combination is hereinafter referred to as including following pharmaceutical composition：(a) change selected from formula (I) compound, formula (II) Close object and formula (III) compound phosphatidyl-inositol 3-kinase or its pharmaceutically-acceptable salts and (b) at least one c-Met by Body tyrosine kinase inhibitor or its pharmaceutically-acceptable salts.

It clearly indicates unless otherwise prescribed or by context or inapplicable, otherwise refers to the treatment combined for the present invention Agent includes the free alkali of compound and all pharmaceutically-acceptable salts of the compound.

In a preferred embodiment of the present invention, the combination includes (a) inhibitors of phosphatidylinositol3 3-kinase Close object A, compound B, compound C or compound D or its pharmaceutically-acceptable salts and the fluoro- N- methyl -4- of (b) 2- [7- quinoline - 6- bases-methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide or its pharmaceutically-acceptable salts.

In another preferred embodiment of the present invention, the combination includes (a) inhibitors of phosphatidylinositol3 3-kinase Compound C or its pharmaceutically-acceptable salts and (b) at least one c-Met inhibitor, selected from the fluoro- N- methyl -4- of 2- [7- quinoline - 6- bases-methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide or its pharmaceutically-acceptable salts.

In another preferred embodiment of the present invention, the combination includes (a) inhibitors of phosphatidylinositol3 3-kinase Compound D or the fluoro- N- methyl -4- of its pharmaceutically-acceptable salts and (b) 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] -2 base of triazine] benzamide or its pharmaceutically-acceptable salts.

According to the present invention, the present invention combines the proliferative diseases for treating object in need.Present invention combination is available In the proliferative diseases for treating object in need, include following pharmaceutical composition by being applied to object：(a) it is a effective amount of Selected from formula (I) compound, formula (II) compound and formula (III) compound phosphatidyl-inositol 3-kinase or its is pharmaceutically acceptable Salt and (b) a effective amount of fluoro- N- methyl -4- of at least one c-Met receptor tyrosine kinase inhibitors such as 2- [7- quinoline -6- Base-methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide or its pharmaceutically-acceptable salts.Preferably, these Therapeutic agent is applied with treating effective dosage, and common advantageous effect can be provided in combination.The application can be separated, same When or sequence apply.

In a preferred embodiment, the proliferative diseases are c-Met dependence proliferative diseases.

The property of the proliferative diseases particularly c-Met dependences proliferative diseases is polyfactorial.In some cases, The different drug of mechanism of action can be combined.However, only considering the different any pharmaceutical composition of binding mode, differ fixed output quota The raw combination with advantageous effects.

It has been found that application present invention combination can be used for object of the treatment with proliferative diseases, particularly lung cancer is (such as non-small Cell lung cancer) or spongioblastoma.In the present invention, generate using present invention combination and more useful treat, such as cooperate with or improve Anti-proliferative effect, such as be related to compared with any single therapy the delay of proliferative diseases progress or be related to gross tumor volume change.

In a preferred embodiment, present invention combination is lost for treating c-Met and/or HGF/c-Met signal paths The proliferative diseases of tune.In one embodiment, which is the imbalance of c-Met and/or HGF/c-Met signal paths Cancer.In another embodiment, the proliferative diseases are the cancers of HGF/c-met gene overexpressions and amplification.

Suitable for the proliferative diseases particularly c-Met dependences proliferative diseases of combined therapy of the present invention, including but not It is limited to cancer, atherosclerosis, pulmonary fibrosis, kidney fibrosis and regeneration, hepatopathy, anaphylactia, inflammation and autoimmunity Property disease, cranial vascular disease, angiocardiopathy and with the relevant illness of organ transplant.

In one embodiment, the proliferative diseases are cancers.Term " cancer " is used to refer to wide spectrum model herein The tumour enclosed, including all entities and Hematological malignancies.The example of this kind of tumour includes but not limited to the benign of lower portion And malignant tumour：Mammary gland, bladder, cervix, extrahepatic bile ducts, Colon and rectum, oesophagus, stomach, head and neck, kidney (such as papillary renal cell Cancer), liver (such as hepatocellular carcinoma), lung (such as Small Cell Lung Cancer and non-small cell lung cancer), nasopharynx, ovary, pancreas, prostate, first shape Gland, endometrium, musculoskeletal system sarcoma (such as osteosarcoma, synovial sarcoma, rhabdomyosarcoma), soft tissue sarcoma (such as MFH/ Fibrosarcoma, leiomyosarcoma, Kaposi sarcoma), Huppert's disease, lymthoma, adult T cell leukemia, acute bone Myelogenous leukemia, chronic myelogenous leukemia, spongioblastoma, astrocytoma, melanoma, celiothelioma and embryonal carcinoma meat Knurl etc..

In yet another embodiment of the present invention, the proliferative diseases are solid tumors.Term " solid tumor " is especially Breast cancer, carcinoma of urinary bladder, oophoroma, colorectal cancer, melanoma, gastric cancer, cervical carcinoma, lung cancer (such as Small Cell Lung Cancer and non-small cell Lung cancer), head and neck cancer, prostate cancer or Kaposi sarcoma.Present invention combination inhibits the growth of solid tumor and liquid tumor.

In yet another embodiment of the present invention, the proliferative diseases are lung cancer (such as non-small cell lung cancer) or plastic Cell plastid knurl.

It should be understood that the present invention includes other embodiment, the above-mentioned proliferative diseases of any of which or cancer are that c-Met is relied on Property proliferative diseases or cancer.

In another embodiment, the present invention relates to be proliferated for treating proliferative diseases particularly c-Met dependences Property disease present invention combination, disease tolerance c-Met receptor tyrosine kinase inhibitors treatments, the fluoro- N- first of particularly 2- Base -4- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine -2 base] benzamide or its is pharmaceutically acceptable Salt.When the term as used herein " tolerance " refers to continued presence c-Met receptor tyrosine kinase inhibitors, previous sensibility tumor Occur after treatment leads to atrophy regrowth or treatment lead to of short duration elimination after occur again.The successful treatment of tolerance tumour can draw Playing such as tumour cell increases, and can cause example the sensibility of novel or previous attempted antineoplaston and/or chemotherapeutics As follow-up death of neoplastic cells and prevented from shifting.

In another embodiment, the present invention relates to for inhibit cancer grow and/or transfer diffusion the present invention combination, Particularly c-Met dependent tumors.Present invention combination is suitable for treatment poor prognosis patient, especially with pulmonary metastasis (such as Non-small cell lung cancer) or spongioblastoma these poor prognosis patient.

In another embodiment, the present invention relates to including following pharmaceutical composition：(a) phosphatidyl-inositol 3-kinase presses down Inhibitor compound C or compound D or its pharmaceutically-acceptable salts and the fluoro- N- of (b) c-Met receptor tyrosine kinase inhibitors 2- Methyl -4- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine -2 base] benzamide or its can pharmaceutically connect By salt, for treating proliferative diseases, particularly c-Met dependences proliferative diseases.Preferably, the proliferative diseases are cancers Disease, most preferably lung cancer (such as non-small cell lung cancer) or spongioblastoma.

In another embodiment, the present invention relates to a pharmaceutical composition, including：(a) phosphatidyl-inositol 3-kinase Inhibitor compound C or its pharmaceutically-acceptable salts and (b) c-Met receptor tyrosine kinase inhibitors PF2341066 are (also referred to as Buddhist nun is replaced for gram azoles) or its pharmaceutically-acceptable salts, for treating proliferative diseases, particularly c-Met dependences proliferative diseases. Preferably, the proliferative diseases are cancers, most preferably spongioblastoma.

On the one hand, the present invention is controlled about a kind of method for treating proliferative diseases including being applied in object in need It can be effectively for the following combination of the amount of proliferative diseases in treatment：(a) selected from formula (I) compound, formula (II) compound and formula (III) phosphatidyl-inositol 3-kinase of compound or its pharmaceutically-acceptable salts and (b) at least one c-Met receptor tyrosines Kinase inhibitor or its pharmaceutically-acceptable salts.

Can be separated to the patient with proliferative diseases particularly lung cancer (such as non-small cell lung cancer) or spongioblastoma, It is administered simultaneously or sequentially including following combination：(a) selected from formula (I) compound, formula (II) compound and formula (III) compound Phosphatidyl-inositol 3-kinase or its pharmaceutically-acceptable salts and (b) at least one c-Met receptor tyrosine kinase inhibitors or its Pharmaceutically-acceptable salts, for treating the proliferative diseases according to the present invention.

In another embodiment, the present invention is about a kind of method for treating proliferative diseases, including in need Object application in the treatment can be effectively for the following combination of the amount of proliferative diseases：(a) inhibitors of phosphatidylinositol3 3-kinase Compound C or compound D or the fluoro- N- methyl of its pharmaceutically-acceptable salts and (b) c-Met receptor tyrosine kinase inhibitors 2-- 4- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine -2 base] benzamide or its pharmaceutically-acceptable salts. The proliferative diseases are preferably c-Met dependence proliferative diseases.The proliferative diseases are preferably cancer, most preferably lung cancer (such as non-small cell lung cancer) or spongioblastoma.

In another embodiment, the present invention is about a kind of method for treating proliferative diseases, including in need Object application in the treatment can be effectively for the following combination of the amount of proliferative diseases：(a) inhibitors of phosphatidylinositol3 3-kinase Compound C or its pharmaceutically-acceptable salts and (b) c-Met receptor tyrosine kinase inhibitors PF2341066 (also referred to as gram azoles For Buddhist nun) or its pharmaceutically-acceptable salts.The proliferative diseases are preferably c-Met dependence proliferative diseases.The proliferative Disease is preferably cancer, most preferably spongioblastoma.

In another embodiment, the present invention inhibits to suffer from the method that shifts formation in cancer object about a kind of, including to Object application in need in the treatment can be effectively for the following combination of the amount of proliferative diseases：(a) selected from formula (I) chemical combination Object, the phosphatidyl-inositol 3-kinase of formula (II) compound and formula (III) compound or its pharmaceutically-acceptable salts and (b) are at least A kind of c-Met receptor tyrosine kinase inhibitors or its pharmaceutically-acceptable salts.The cancer is preferably c-Met dependence cancers Disease.Suitable cancer be more than be described in detail and be incorporated herein by reference in embodiments those.

In a preferred embodiment, this method and combination can be used to treat lung cancer (such as non-small cell lung cancer) or plastic Cell plastid knurl.

On the one hand, the present invention in treatment proliferative diseases and/or prepares treatment proliferative diseases medicine about present invention combination Purposes in object.In a preferred embodiment, the proliferative diseases are c-Met dependence proliferative diseases.At one In preferred embodiment, the proliferative diseases are cancers.Suitable proliferative diseases and/or cancer are in above embodiment party Those for being described in detail and being incorporated herein by reference in formula.

In one embodiment, the present invention is about selected from formula (I) compound (such as compound A or B), formula (II) compound The inhibitors of phosphatidylinositol3 3-kinase of (such as compound C) and formula (III) compound (such as compound D) or its is pharmaceutically acceptable The combination of salt and at least one c-Met receptor tyrosine kinase inhibitors or its pharmaceutically-acceptable salts is preparing the purposes treated Purposes in proliferative diseases drug.In a preferred embodiment, the proliferative diseases are c-Met dependence proliferatives Disease.In a preferred embodiment, the proliferative diseases are cancers, particularly lung cancer (such as non-small cell lung cancer) or into Spongiocytoma.

In another embodiment, the present invention about inhibitors of phosphatidylinositol3 3-kinase compound C or compound D or Its pharmaceutically-acceptable salts and the fluoro- N- methyl -4- of c-Met receptor tyrosine kinase inhibitors 2- [7- quinoline -6- bases-methyl) - - 2 base of imidazo [1,2-b] [1,2,4] triazine] combinations of benzamide or its pharmaceutically-acceptable salts treats proliferative preparing Purposes in disease medicament.In a preferred embodiment, the proliferative diseases are c-Met dependence proliferative diseases. In a preferred embodiment, the proliferative diseases are cancers, particularly lung cancer (such as non-small cell lung cancer) or collagen Cytoma.

In another embodiment, the present invention about inhibitors of phosphatidylinositol3 3-kinase compound C or its pharmaceutically Acceptable salt and c-Met receptor tyrosine kinase inhibitors PF2341066 (also referred to as (R)-3-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridin-2-amine) or its can pharmaceutically connect The purposes in treating proliferative diseases drug is being prepared by the combination of salt.In a preferred embodiment, the proliferative disease Disease is c-Met dependence proliferative diseases.In a preferred embodiment, the c-Met dependences proliferative diseases are cancers Disease, particularly spongioblastoma.

On the one hand, the present invention suffers from shifting to be formed and/or prepared in cancer object and inhibits to suffer from cancer about present invention combination in inhibition Purposes in the drug that transfer is formed in object.In a preferred embodiment, the cancer is c-Met dependence cancers.

Advantageous effect, such as the single treatment with individual treatment agent in the combination may be not only generated using present invention combination The therapeutic effect of method compares such as synergistic therapeutic effect, such as is related to alleviating, postpones symptom development or inhibit symptom, but also generate Surprising advantageous effect, the monotherapy of rem is compared as used in being combined with using a kind of present invention, side effect Less, quality of life improves or incidence reduces.

Another benefit is that the therapeutic agent that combine of the present invention of lower dosage can be used, such as dosage not only usual smaller, and And frequency of administration is lower or can occur to reduce the side effect observed during a kind of therapeutic agent of separate administration.This with it is to be treated The hope of patient is consistent with demand.

It can be shown by established test model, present invention combination generates advantageous effect described previously herein.Ability Field technique personnel are entirely capable of selection relevant test model to prove the advantageous effect.For example, the pharmacology that the present invention combines is lived Property can be proven during clinical research or in vitro test, substantially as described below.

Suitable the clinical research in particular, for example open label in proliferative diseases patient, dose escalation study, it is special It is not c-Met dependence proliferative diseases, especially lung cancer (such as non-small cell lung cancer) or spongioblastoma.These researchs are special The concertedness of therapeutic agent that of the invention Zheng Ming not combine.The result of these researchs well known by persons skilled in the art can directly be passed through Determine the advantageous effect to c-Met dependence proliferative diseases.These researchs are especially suitable for comparing the single of any therapeutic agent The effect that therapy and the present invention combine.In one embodiment, increase selected from formula (I) compound (such as compound A or B), formula (II) dosage of the phosphoinositide-3 kinase of compound (such as compound C) and formula (III) compound (such as compound D), Until reach maximum tolerated dose, and the fluoro- N- methyl -4- of c-Met receptor tyrosine kinase inhibitors such as 2- [7- quinoline -6- bases - Methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide applied with fixed dosage.Alternatively, selected from formula (I) chemical combination The phosphatidyl-4 of object (such as compound A or B), formula (II) compound (such as compound C) or formula (III) compound (such as compound D) 3 kinase inhibitor of alcohol can be applied with fixed dosage, and the fluoro- N- methyl -4- of c-Met receptor tyrosine kinase inhibitors such as 2- The dosage of [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine -2 base] benzamide can be incremented by.Each patient can To receive the dosage of phosphoinositide-3 kinase daily or intermittently.The effect for the treatment of, can be in this kind of research In determine, such as after 12,18 or 24 weeks, by every 6 weeks assess symptom score determine.

To determine the cooperative interaction between one or more components, optimized scope and each group for the effect Divide absolute dose ranges can be by clearly being surveyed using component to patient in need for the treatment of with different w/w proportional regions and dosage Amount.For people, completing the complexity of the clinical research of patient and cost may cause this test form as main concertedness mould The purposes of type is unrealistic.However, observe that synergisticing performance is present in the effect other objects described herein In a species Kind and animal model play indication, and to measure synergy, and the result of this kind of research passes through using pharmacokinetics/pharmacodynamics Method can be used for predicting effective dose proportional region, absolute dosages and the plasma concentration needed for other species.Tumor model with The correlation established seen in human body between effect show concertedness in animal can for example, by heteroplastic transplantation model or Suitable cell line proves.

The combination of inhibitors of phosphatidylinositol3 3-kinase can be used in the method for the present invention, is configured to comprising one or more pharmacy The pharmaceutical composition of upper acceptable carriers.

On the one hand, the present invention is provided effectively is directed to proliferative diseases particularly c-Met dependences comprising common in the treatment The present invention combination of the amount of proliferative diseases and the pharmaceutical composition of one or more pharmaceutically acceptable carriers.The pharmaceutical composition Object can be made of therapeutic agent (a) and (b), be applied as fixed Combination with single formulation or unit dosage forms by any appropriate approach For patient.Alternatively, the therapeutic agent such as inhibitors of phosphatidylinositol3 3-kinase and c-Met receptor tyrosine kinase inhibitors or Its pharmaceutically-acceptable salts is all applied to patient as non-fixed combinations with single pharmaceutical composition or preparation or unit dosage forms, and And simultaneously, synchronous or sequence application and limited without specific time.

In a preferred embodiment, the present invention, which provides, is separately contained in treatment above jointly effectively for proliferative diseases The particularly pharmaceutical composition of the therapeutic agent (a) of the amount of c-Met dependences proliferative diseases and therapeutic agent (b).The therapeutic agent Synchronous but separate administration or sequence are applied to object in need.

For the therapeutic agent of separate administration or with fixed Combination (the single galenica combined comprising the present invention) application The pharmaceutical composition of therapeutic agent can prepare in a way known, and intestines is suitble to apply such as oral or per rectum and stomach It is parenteral to be applied to object including people (warm-blooded animal), the independent medicine as described above of at least one comprising therapeutically effective amount Active therapeutic agent or the one or more pharmaceutically acceptable carriers of joint are managed, are especially suitable for intestines or parenteral administration.

The new pharmaceutical composition can include about 0.1%- about 99.9%, the therapeutic agent of preferably from about 1%- about 60%.

For intestines application or parenteral administration, include fixed Combination or non-solid for the pharmaceutical composition of combination therapy Fixed combination, for example, by using unit dosage forms, such as sugar coated tablet, tablet, capsule or suppository or ampoule bottle.Unless otherwise indicated, Prepare in a way known, for example, by those skilled in the art know that a variety of conventional mixing, crush, be granulated, Sugar coating, dissolving, lyophilized technique or manufacturing technology.It should be understood that the unit content of therapeutic agent contained by each dosage form individual dose is in itself It is not required to form effective quantity, because required effective quantity can be reached by the multiple dosage units of application.

Unit dosage forms containing reagent combination or the individual reagent of reagent combination, which can be used, to be packaged in capsule such as gelatine capsule Form of minitablets.For this purpose, the gelatine capsule used in pharmaceutical preparation can be used, it is such as obtained from being known as Pfizer (Pfizer) The hard gelatin capsule of CAPSUGEL.

The unit dosage forms of the present invention can optionally further comprise the additional conventional carrier or excipient for drug.The load Body example include but not limited to disintegrant, adhesive, lubricant, glidant, stabilizer and filler, diluent, colorant, Flavoring agent and preservative.Those of ordinary skill in the art dosage form property can select one kind according to needed for specific by routine experiment Or a variety of above-mentioned carriers, without any undue burden.The amount of each carrier used can change in the normal ranges of this field.It is following The bibliography being incorporated herein by reference of text discloses the technology and excipient for preparing peroral dosage form.Referring to《It is medicinal Excipient Handbook》(The Handbook of Pharmaceutical Excipients), the 4th edition, the volumes such as Rowe, U.S.'s medicine Learn (American Pharmaceuticals Association) (2003)；With《Remington：Pharmaceutical science and practice》 (Remington:The Science and Practice of Pharmacy), the 20th edition, Gennaro is compiled, Donald Lippincott prestige Lian Si Louis Wilkins publishing company (Lippincott Williams Wilkins) (2003).

By by one or more conventional carriers before melt granulation or period is included in original mixture or by oral One or more conventional carriers and particle are combined in dosage form, these optional additional conventional carriers can be included in peroral dosage form.Rear In the embodiment in face, the mixture of merging can further be mixed for example, by V-Mixer, then suppress or be shaped to piece Agent, such as individual layer tablet, by encapsulated or it is filled into deck.

The example of pharmaceutically acceptable disintegrant includes but not limited to starch；Clay；Cellulose；Alginates；Natural gum；Crosslinking Polymer, such as crosslinked polyvinylpyrrolidone or crospovidone, such as ISP (International Specialty Products) the POLYPLASDONE XL of (New Jersey Wei grace)；Croscarmellose sodium, such as the AC-D1- of FMC SOL；With cross-linked carboxymethyl cellulose calcium；Soybean polyoses；And guar gum.The existing amount of the disintegrant can be about 0%- about 10% composition weight.In one embodiment, the existing amount of the disintegrant is the composition weight of about 0.1%- about 5% Amount.

The example of pharmaceutically acceptable adhesive includes but not limited to starch；Cellulose and its derivates, such as crystallite are fine Dimension element such as the AVICEL PH of FMC (philadelphia, pa), hydroxypropyl cellulose, hydroxyethyl cellulose and hydroxypropyl methyl Cellulose, such as the METHOCEL of Dow Chemical's (Dow Chemical Corp.) (available)；Sucrose；Grape Sugar；Corn syrup；Polysaccharide and gelatin.The existing amount of described adhesive can be the composition weight of about 0%- about 50%, example Such as the composition weight of 2%-20%.

The example of pharmaceutically acceptable lubricant and pharmaceutically acceptable glidant includes but not limited to silica gel, three silicic acid Magnesium, starch, talcum powder, calcium phosphate,tribasic, magnesium stearate, aluminum stearate, calcium stearate, magnesium carbonate, magnesia, poly- second two Alcohol, powdered cellulose and microcrystalline cellulose.The existing amount of the lubricant can be the composition weight of about 0%- about 10%. In one embodiment, the existing amount of the lubricant can be the composition weight of about 0.1%- about 1.5%.The glidant Existing amount can be the composition weight of about 0.1%- about 10%.

The example of pharmaceutically acceptable filler and pharmaceutically acceptable diluent includes but not limited to Icing Sugar, compressible Sugar, dextrates, dextrin, glucose, lactose, mannitol, microcrystalline cellulose, powdered cellulose, sorbierite, sucrose and cunning Mountain flour.For example, the filler and/or the existing amount of diluent can be the composition weights of about 0%- about 80%.

In one embodiment, described pharmaceutical composition includes common effectively special for proliferative diseases in the treatment It is the present invention combination of the amount of c-Met dependence proliferative diseases and one or more pharmaceutically acceptable carriers, wherein phosphatide Acyl inositol 3-kinase inhibitor is selected from compound A, compound B, compound C and compound D or its pharmaceutically-acceptable salts.

In another embodiment, described pharmaceutical composition includes common effectively for proliferative diseases in the treatment The present invention combination of amount and one or more pharmaceutically acceptable carriers, wherein c-Met inhibitor is the fluoro- N- methyl -4- [7- of 2- Quinoline -6- bases-methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide or its pharmaceutically-acceptable salts.

In another embodiment, described pharmaceutical composition includes common effectively for proliferative diseases in the treatment The present invention combination of amount and one or more pharmaceutically acceptable carriers, wherein being of inhibitors of phosphatidylinositol3 3-kinase Close object C, compound D or its pharmaceutically-acceptable salts, the c-Met inhibitor be the fluoro- N- methyl -4- of 2- [7- quinoline -6- bases - Methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide or its pharmaceutically-acceptable salts.

In another embodiment, described pharmaceutical composition includes common effectively for proliferative diseases in the treatment The present invention combination of amount and one or more pharmaceutically acceptable carriers, wherein being of inhibitors of phosphatidylinositol3 3-kinase Close object C, compound D or its pharmaceutically-acceptable salts, the c-Met inhibitor be PF2341066 (also referred to as gram azoles replaces Buddhist nun) or Its pharmaceutically-acceptable salts.

According to the present invention, each therapeutic agent that the present invention of therapeutically effective amount combines can while or sequentially be applied in any order With the component can be applied separately or as fixed Combination.For example, it may include according to the method for present invention treatment proliferative diseases (i) using the first therapeutic agent (a) and (ii) of free form or pharmaceutically-acceptable salts form using free form or pharmaceutically The therapeutic agent (b) of pharmaceutically acceptable salt, while or sequentially apply in any order, using combination therapy effective quantity, preferably cooperate with Effective quantity, such as the daily or intermittent doses corresponding to amount described herein.The individual treatment agent that the present invention combines can treat Different time separate administration in process or synchronized with separated or single combining form is applied.In addition, term administering " also wrap It includes using the therapeutic agent prodrug for being converted to therapeutic agent in vivo.Therefore, the present invention should be understood to cover all such while or hand over For the scheme for the treatment of, term administering " also should correspondingly it explain.

The effective dose of each therapeutic agent can be according to specific compound used or pharmaceutical composition, application used in present invention combination Pattern treats illness, treats the seriousness of illness and change.Therefore, the dosage that combines of the present invention according to it is a variety of because Element selection, kidney and liver function including administration method and patient.The clinician or doctor for grasping ordinary skill can easily determine With the prescription for issuing a effective amount of single therapy agent needed for alleviation, counteracting or retardance disease progression.

Other compounds that the effective dose of each therapeutic agent may need a kind of compound to compare the combination are more frequently applied. Therefore, to allow appropriate application, the drug products of packaging may include one or more dosage forms comprising compound combination and one Kind or a variety of dosage forms comprising one of compound combination and not comprising the other compounds of the combination.

For the therapeutic agent that combines of the present invention as single medicine commercial form in use, its dosage and administration mode should be with The information that each marketed drugs package insert is provided is consistent, unless mentioned otherwise herein.

It is to be administered in the compound of formula I especially compound A of the object in need such as from about people of 70kg weight or its pharmacy The dosage of upper acceptable salt preferably for each person every day about 3mg- about 5g, more preferably from about 10mg- about 1.5g, more preferably from about 100mg-about 1200mg, most preferably from about 100mg-about 1000mg, are preferably divided into 1-3 single dose, can be for example with same size.

Formula II compound especially compound C is preferably daily with the dosage range of about 0.001-1000mg/kg weight daily Using more preferable 1.0-30mg/kg weight.In a preferred embodiment, the compound of formula I especially dosage of compound C Range is about 10mg- about 2000mg for each person every day.

Formula III compound especially compound D is preferably with the dosage range of about 0.05-50mg/kg recipient's weight daily Application daily, preferably from about 0.1-25mg/kg/, more preferably from about 0.5-10mg/kg/.Therefore, for the people of 70kg applies, Dosage range is most preferably daily about 35-700mg.

The fluoro- N- methyl -4- of c-Met receptor tyrosine kinase inhibitors especially 2- [7- quinoline -6- bases-methyl) - - 2 base of imidazo [1,2-b] [1,2,4] triazine] benzamide is preferably with the agent of about 0.01mg/kg- about 100mg/kg weight daily Amount application daily.Therefore, for the people of 70kg applies, dosage range is most preferably daily about 0.7mg-7g, more preferably daily About 50mg-300mg.

Optimal dosage for treating each therapeutic agent of proliferative diseases can be empirically for each individual with known method It determines, and depending on many factors, including but not limited to progression of disease degree；The age of individual, weight, general health, gender And diet；Administration time and approach；The other medicines that individual is taken.Optimal dosage can use routine test well known in the art and mistake Journey determines.

Can combine with carrier material each therapeutic agent that single formulation is made amount can according to treat individual and specific application Pattern and change.In some embodiments, the unit dosage forms of the combination containing reagent described herein can separately be applied comprising the reagent Each combination amount of reagent that used time usually applies.

Dose frequency can change according to compound used therefor and particular condition to be treated.It is generally preferable that using being enough to carry For the minimum dose effectively treated.It is general to be monitored with being suitble to treat the experiment well known within the skill of those ordinarily skilled of illness The treatment validity of patient.

In one embodiment, the present invention provides a kind of pharmaceutical composition, including therapeutic compounds A or B or its pharmacy Upper acceptable salt and c-Met receptor tyrosine kinase inhibitors, and the fluoro- N- methyl -4- of preferably 2- [7- quinoline -6- bases-methyl)-miaow Azoles simultaneously -2 base of [1,2-b] [1,2,4] triazine] benzamide or its pharmaceutically-acceptable salts, it can be with disclosed herein group It closes, pharmaceutical composition and dosage form exists, and proportional region is respectively daily 20:1-2:1, more preferable 20:1-5:1.

In a preferred embodiment, the present invention provides a kind of pharmaceutical composition, including therapeutic compounds C or its pharmacy Upper acceptable salt and c-Met receptor tyrosine kinase inhibitors, and the fluoro- N- methyl -4- of preferably 2- [7- quinoline -6- bases-methyl)-miaow Azoles simultaneously -2 base of [1,2-b] [1,2,4] triazine] benzamide or its pharmaceutically-acceptable salts, it is ranging from daily with dose ratio 1:1-1:4, more preferably daily 1:1 or 1:2 exist.

In a preferred embodiment, the present invention provides a kind of pharmaceutical composition, including therapeutic compounds D or its pharmacy Upper acceptable salt and c-Met receptor tyrosine kinase inhibitors, and the fluoro- N- methyl -4- of preferably 2- [7- quinoline -6- bases-methyl)-miaow Azoles simultaneously -2 base of [1,2-b] [1,2,4] triazine] benzamide or its pharmaceutically-acceptable salts, be respectively with dose ratio range Daily 8:1-1:1, more preferable 4:1-2:1-1:1 exists.

It generates effect and the best proportion of avirulent medical compounds, separate and unitized dose and concentration are based on controlling Dynamics of the agent to the availability of target site is treated, and is measured with methods known to those skilled in the art.

In addition, the present invention is provided comprising present invention combination as the commercial package of therapeutic agent and is being treated for it The specification simultaneously, separately or sequentially applied in proliferative diseases.In a preferred embodiment, the proliferative diseases are C-Met dependence proliferative diseases.In a preferred embodiment, the proliferative diseases are lung cancer (such as non-small cell lungs Cancer) or spongioblastoma.

The following example illustrates foregoing invention；But it is not intended to limit invention scope in any way.Pharmaceutical composition of the present invention Advantageous effect other test models can also be determined known to various equivalent modifications.

As described herein, the effectiveness that the present invention combines can be tested, be demonstrate,proved in animal test model and clinical research in vitro It is bright.For example, the effectiveness of formula (I) compound according to the present invention can be proved according to following methods：

Embodiment 1：The combination of compound A is used in non-small cell lung cancer

Material and method

PI3K inhibitor (compound A) and the fluoro- N- methyl -4- of c-Met receptor tyrosine kinase inhibitors 2- [7- quinoline - 6- bases-methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide combined evaluation in non-small cell lung cancer model. The Compound Stock solution of two kinds of compounds is prepared separately in DMSO.Compound with the serial dilution of Tecan dispensers with cover~ 1000x concentration ranges.It is as follows for the maximum concentration of experiment：A=2.7 μM of compound and the fluoro- N- methyl -4- of 2- [7- quinoline -6- Base-methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide=0.27 μM.Single agents and combination are all with multiple Concentration determination, together with control and a selfing.

Cell culture and vigor measure

American Type Culture Collection (American Type Cell are purchased from for the cell line of this research Collection), including Non-small cell lung carcinoma cell line EBC-1 (its with c-Met expand), NCI-H1993, (it has expansion The c-MET of increasing), NCI-H1648 (its with c-Met expand), NCI-H1573 (it is expanded with c-Met and KRAS mutation) and HCC2935 (EGFR mutation).According to the explanation of supplier, all cell lines are cultivated in respective culture medium.

In order to assess combined effect, cell is inoculated in 384 orifice plates with 500 cells/wells and is incubated overnight.The chemical combination The content pre-dilution 1 of owner's plate:200 (1 μ L compound solutions cell RPMI-160 trainings of the 200 μ L containing 10% fetal calf serum Support base), this pre-dilution liquid of 5 μ L is then shifted to the cell plates containing 20 μ L cell culture mediums, to reach target end compound concentration And 0.09% supporting agent (DMSO) concentration.

By quantitative cellular ATP levels (CellTiterGlo, Pu Luomaige (Promega)) in 37 DEG C/5%CO₂It is incubated Evaluated after 72 hours single agents with and combinations thereof to the effect of cell viability, use 25 μ L reagents/hole and n=2 repeat plates/item Part.Similary cell quantity/the vigor evaluated when compound is added simultaneously is used to estimate cell line population doubling time.

For untreated and through handling horizontal U and T, inhibit to be calculated with Formulas I=1-T/U, just completely inhibit for its range For 0%-100%.Work as T<U₀When, " growth inhibition " GI=1-(T-U₀)/V₀, and in addition in the case of 1-(T-U₀)/(U-U₀), wherein U₀Untreated level when be the time being zero.0th, 100% and 200% GI levels correspond to inactivation, cell inhibits and cell toxicant Property compound.

Compound activity, concertedness and selectivity calculate

By being fitted form I=I_maxC^α/[C^α+EC₅₀ ^α] S-shaped Xi Er functions come identify the single agents of each compound live Property, wherein C is concentration, EC₅₀It is inflection point, and α is S-shaped variation.Calculate IC₅₀Intersection point, wherein matched curve reach 50% inhibition.

For combination, concertedness is calculated as Loewe additive models, and (" drug is in itself " generated by increase effective dose is pre- Phase).Each dosage is calculated to C_X,YLoewe be expected (Loewe S., Ergebn Physiol27:47-187 (1928)), this is logical It crosses to find and inhibits I_Loewe, so as to (C_X/IC_X)+(C_Y/IC_Y)=1 and IC_X,YIt is the I for being fitted single agents curve_LoewePlace Effective concentration uses numerical optimization [Berenbaum MC, J Theor Biol114:413–431(1985)]." collaboration scoring " S It is calculated as below：To I in all test unitized dose points_DataWith I_LoeweBetween difference be averaged, exclude the highest of dose matrix Concentration point, most probable are controlled by drug off-target effects.GI_maxIt is maximum growth inhibition (the exclusion maximum dose level of matrix It is right), the measurement as overall combination effect.

Selective collaboration is evaluated by carrying out collaboration scoring to combination in all test cell systems relatively.Single cell system In " selectivity scoring " of the combination be calculated as SS=abs (S) * GI_max* (S- μ)/σ, wherein μ is intermediate value and σ is all cells The median absolute deviation of S in system.This weight Z score protrudes the combination that cooperates with extremely in cell line and itself is effective and cooperates with 's.

All calculate is carried out with Chalice softwares (CombinatoRx, Massachusetts Cambridge).In this experiment, GImax Data are understood with following scale：(a) GImax=" 0 " represents unrestraint, and GImax=about " 1 " represents 100% inhibition and static state, with And GImax=2 represents that 200% inhibits the overall elimination or dead with cancer cell.Using this program, the combination of maximum concentration is excluded Effect (GImax) is as follows：

In this experiment, concertedness scoring (SS) description below：(a) SS=" 0 " represents collaboration, and (b) SS=" 0.1 " represents high The negative value of collaboration and (c) SS=" -0.01 to -0.07 " represent additive property.The selectivity collaboration scoring of acquisition should be combined (excluding maximum concentration) is as follows：

Embodiment 2：The combination of compound C is used in non-small cell lung cancer

According to the experimental arrangement described in example 1 above, PI3K inhibitor chemical combination is assessed in non-small cell lung cancer model The object C and fluoro- N- methyl -4- of c-Met receptor tyrosine kinase inhibitors 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] -2 base of triazine] benzamide combination the effect of and concertedness.Compound is with the serial dilution of Tecan dispensers to cover ~1000x concentration ranges.It is as follows for the maximum concentration of experiment：C=11 μM of compound and the fluoro- N- methyl -4- of 2- [7- quinoline - 6- bases-methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide=0.27 μM.Single agents and combination are all with more A concentration determination, together with control and a selfing.

All calculate is carried out with Chalice softwares (CombinatoRx, Massachusetts Cambridge).Using this program, exclude The combined effectiveness (GImax) of maximum concentration is as follows：

In addition, the combination selectively collaboration scoring for excluding maximum concentration is as follows：

Embodiment 3：The combination of compound D is used in non-small cell lung cancer

According to the experimental arrangement described in example 1 above, PI3K inhibitor chemical combination is assessed in non-small cell lung cancer model The object D and fluoro- N- methyl -4- of c-Met receptor tyrosine kinase inhibitors 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] -2 base of triazine] benzamide combination the effect of and concertedness.Compound is with the serial dilution of Tecan dispensers to cover ~1000x concentration ranges.It is as follows for the maximum concentration of experiment：D=11 μM of compound and the fluoro- N- methyl -4- of 2- [7- quinoline - 6- bases-methyl) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide=0.27 μM.For experiment minimum concentration such as Under：D=0.15 μM of compound and the fluoro- N- methyl -4- of 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] three - 2 base of piperazine] benzamide=0.0003 μM.Single agents and combination are all with multiple concentration determinations, together with control and a selfing.

All calculate is carried out with Chalice softwares (CombinatoRx, Massachusetts Cambridge).Using this program, exclude The combined effectiveness (GImax) of maximum concentration is as follows：

In addition, the combination selectively collaboration scoring for excluding maximum concentration is as follows：

Embodiment 4：The combination of compound C is used in spongioblastoma

Method

Assess PI3K inhibitor compound C and c-Met receptor tyrosines in combination in spongioblastoma model The fluoro- N- methyl -4- of kinase inhibitor 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine -2 base] benzene first Amide.The fluoro- N- methyl -4- of compound C (10mM) and 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] three - 2 base of piperazine] benzamide (10mM) is dissolved in DMSO, and is preserved in -20 DEG C of deciles.

Cell culture and cell viability detection

People's spongioblastoma cell line U-87MG (PTEN mutant and HGF expression system), H4 (PTEN mutant and HGF Expression system), A172 (the low PTEN mutational cell lines of HGF expressions) and LN-229 (PTEN wild types and HGF is low) be purchased from U.S. Type Culture Collection institute of state is simultaneously maintained at 37 DEG C, 5%CO₂Humidified incubator, in the culture medium in recommendation.All 4 kinds Cell line expresses detectable MET in mRNA level in-site.

Cell is passaged twice a week, and culture medium was replaced per 2-3 days.Cell viability is detected, cell TryPLE Express carries out trypsin treatment and is inoculated into 96 hole blackboard of clear bottom (Coase tower (Costar), #3904) in triplicate, Can be adherent overnight, then it is incubated 72 hours with the inhibitor of various concentration or reagent combination.

Cell viability is measured by measuring cellular ATP content, is used(CTG) luminescent cell viability examination (Pu Luomaige).By each single agents and combined treatment cell with compareing or being made comparisons with the cell of isometric medium treatment. At the end of compound processing isometric CTG reagents are added in each hole and in Envision plate readers (PerkinElmer (Perkin Elmer it records and shines on)).The luminous signal value (response) for reducing and enhancing is calculated relative to the cell of untreated (control).

The method for calculating combined effect

In order to assess the antiproliferative activity of the PI3K inhibitor and the Cmet inhibitors and identification in a manner of unbiased Synergy under be possible to concentration is studied with " dose matrix ".This employ it is possible that arrangement serial dilution Single agents and " dose matrix ", are made up of：

Compound C receives 5 dosage 2X serial dilutions, and high dose is 2.4 μM and low dosage is about 150nM

This inhibitors of c-met receives 5 dosage 2X serial dilutions, and high dose is 1 μM and low dosage is about 12nM

Cooperative interaction (being analyzed with Chalice softwares [CombinatoRx, Massachusetts Cambridge]) is calculated as below： Compare the reaction from combination to react with what independent reagent acted on, reference model is superimposed for drug dosage itself.Dosage is superimposed Deviation can numerically be evaluated with combinatorial index (CI), the overall strength of quantita-tive combinations effect.This calculating (is substantially volume Score) it is as follows：

V_HSA=Σ_X,Y lnf_X lnf_Y(I_Data–I_HSA)

In addition, calculating the CI between data and highest single agents surface, single agents dilution gfactor is standardized (Lehar J etc. (2009), " synergistic pharmaceutical combination often improves treatment Correlation selection (Synergistic drug combinations tend to improve therapeutically relevant selectivity)”,Nature Biotechnology 27:659-66(2009))。

Data analysis

Data assessment and graphic hotsopt are carried out with Microsoft Excel softwares and Chalice softwares.

As a result

Assess the fluoro- N- methyl -4- of compound C and 2- [7- quinoline -6- bases-first as described above in " dose matrix " scheme Base) -2 base of-imidazo [1,2-b] [1,2,4] triazine] benzamide effect.All 4 kinds of cells fluoro- N- of compound C and 2- Methyl -4- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine -2 base] benzamide is in 96 orifice plates Reason.Cell viability CellTiter-Glo test measurements and % inhibit data to be numerically shown as 6X6 dosage lattice.This paper's Fig. 1-4 provides the result from the above process and summarizes.Each data point represents the average data from 3 holes, and chromatography also represents suppression System is horizontal.Runic rectangle protrudes region of the combination effect higher than the single agents of same dose.

The suppression percentage of each dosage lattice is as shown in Fig. 1-4 of this paper.Compound C shows dense in all 4 kinds of cell lines Dependence antiproliferative activity is spent, relatively low, the fluoro- N- methyl -4- of 2- of activity in a cell line, that is, PTEN wild types (LN229) [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine -2 base] benzamide is in 2 HGF high cell lines Appropriateness activity, complete deactivation in (A172 and LN229) 2 another are only shown in (U87MG and H4).During combination, H4 and U87-MG are Observe concertedness, but A172 and LN229 do not have, this is pressed down by the growth of each single agents in outburst area and equivalent line chart System increases to prove, as shown in Fig. 1-4 of this paper.

This is statistics indicate that combination collaboration inhibits the growth of spongioblastoma cell line subgroup, PTEN mutant and HGF high Cell line is apparently more sensitive to each single agents, and combines and cause more significant growth inhibition.

Embodiment 5：The combination of compound C is used in spongioblastoma heteroplastic transplantation model

This experiment in, U-87MG people's spongioblastoma heteroplastic transplantation model for evaluate PI3K inhibitor compounds C and The fluoro- N- methyl -4- of c-Met inhibitor 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine -2 base] benzene first Amide is as single agents or the antitumor efficacy of combination.It is reported that the U-87MG tumours obtained from National Cancer Institute Cell with regard to CDKN2A, CDKN2C, CDKN2a and PTEN be potentially carcinogenic variant for be homozygous；MET, PIK3CA or other Mutation is not detected in 58 genes frequently changed in neoplastic cell；It is expressed with HGF.

Mouse 17.7 and 35.3mg/kg c-Met inhibitor and 32.7mg/kg compound C monotherapies and c-Met Inhibitor/compound C duplexs therapy (17.7:32.7) daily treatment, continues 21 days.It is 58.8 that research, which includes dose ratio,: C-Met inhibitor/compound C in combination of 5.4mg/kg.Control mice receives 2 kinds of supporting agents, with Temozolomide side before standard clinical Case monitoring tumor model performance.The oral medication of nude mouse with established tumour starts from the 1st day (D1).When tumour reaches To scheduled 2000mm³Terminal volume or to D60 (whichever first occurs), to each euthanizing animals.Research is at D51 ends Only.Short term efficacy is measured according to the average and median tumor volume change of D13, and D13 is to be exited in any group more than 50% mouse Last day before research.Time of total effect according to needed for tumour progression to volume terminal and according to D51 survival rates with disappear Move back rate measure.This report describes the method and result of U87MG-e326 tumor growth delays experiment.

Mouse

In the D1 of research, female nude mice (nu/nu, Charles River (Charles River)) is 10 week old, weight range For 15.5-27.3g.Animal can free water (reverse osmosis, 1ppm Cl), NIH 31Modified and are freely eaten Irradiated Lab Diet (improvement and irradiation experiment room diet)The diet is by 18.0% crude protein, 5.0% thick fat Fat and 5.0% crude fibre composition.Mouse feeder irradiated Enrich-o ' cobs in static state declines isolator^TMIt pads in laboratory On material, if 20-22 DEG C (68-72 °F) and 40-60% humidity and 12 small time cycles.

Tumour transplatation and measurement

U-87MG tumor cell lines are obtained from American Type Culture Collection and pass through continuously to be implanted in nude mouse and maintain. By tumor fragment (1mm³) subcutaneous transplantation is to the right side oxter of each test mice.Tumour slide calliper rule are measured with 2 dimensions so as to supervise Control growth, when its average external volume is close to 100 required -150mm³Range.With mm³The tumor size of meter is calculated as below：Gross tumor volume =(width²X length)/2, wherein width and length is tumour measurement (in terms of mm).Tumor weight can be used it is assumed hereinafter that estimating： 1mg is equal to 1mm³Gross tumor volume.14 days after tumor cell transplantation, in the D1 of research, individual tumors volume is 63-196mm³It is small Mouse is divided into 7 groups, and every group of mean tumour volume is 122-127mm³。

Test specimen

The fluoro- N- methyl -4- of c-Met inhibitor 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine - 2 bases] benzamide (dihydrochloride, 84.98% free alkali) is in 4 DEG C of preservations and with 2X final concentrations addition deionized water, with ultrasound Wave processing is until dissolving.Add in isometric 0.5% methylcellulose and 0.1% for being dissolved in deionized water80 with offer Be dissolved in the 5.884mg/mL of 0.25% methylcellulose using solution and being dissolved in the 0.05% of deionized water80 (carry Agent 1).This solvent portions supporting agent 1 is diluted applies solution with 3.53 and 1.765mg/mL of offer.

Compound C (hydrochloride, 91.85% free alkali), which is preserved and added at -20 DEG C, is dissolved in the 1% of deionized water80, ultrasonication obtains the uniform suspension of 2X final concentrations.Add in the isometric 1% methyl fibre for being dissolved in deionized water Dimension element stirs mixture and carries out the opaque colourless 3.266mg/mL that ultrasonication acquisition is dissolved in 0.5% methylcellulose Using suspension and being dissolved in the 0.5% of deionized water80 (supporting agents 2).It is outstanding that part first is diluted by using supporting agent 2 Liquid come obtain 0.544mg/mL apply suspension.

By be suspended from deionized water and 4 DEG C preservation, once prepare Temozolomide (Schering Corp (Schering Corporation), 100mg capsules, lot number IRSA001).

Treatment plan

Test reagent and supporting agent are all applied by administration by gavage once a day, continuous 21 days (qd × 21).In combination therapy, change Object A is closed to apply in 60 minutes after c-Met inhibitor.Temozolomide ketone once a day apply by administration by gavage, continuous 5 days (qd × 5). In all groups, the administered volume (0.2mL/20g mouse) of 10mL/kg is scaled according to each animal weight that administration is surveyed day, in addition to Continue the weekend of previous BW.

7 groups of nude mouses (n=10/ groups) are treated as follows.1 mouse of group receives supporting agent 1 and 2, and the control as all analyses. In D7, this group uses unintentionally IgG negative control antibody administrations；Think that this error does not influence control tumor growth.Group 2 and 3 connects By c-Met monotherapies, respectively 17.7 and 35.3mg/kg (is equal to 15 and 30mg/kg free alkalis).Group 4 receives 32.7mg/ The compound C monotherapies of kg (30mg/kg free alkalis).Group 5 receives 17.7mg/kg c-Met inhibitor and 32.7mg/kgization Close the combination of object C.Group 6 receives 58.8mg/kg c-Met inhibitor and 5.4mg/kg compounds C (is respectively equivalent to 50 and 5mg/ Kg free alkalis) combination.Group 7 i.e. reference group receives the Temozolomide monotherapy of 100mg/kg.

Tumor growth inhibition

Short term efficacy is measured in D13.Before D13, when gross tumor volume summation develops to terminal, one only has the group 1 of dual tumour In animal exit.D13 data include the gross tumor volume that this animal finally records.Each animal is measured in D1 (using starting) and end Tumor volume difference terminal between point day.For each treatment group, the reaction of terminal day passes through the calculating of one of following relationship：

Wherein

Δ T=(treatment group is in the mean tumour volume of terminal day)-(treatment group is in the mean tumour volume of D1),

Δ C=(control group is in the mean tumour volume of terminal day)-(control group is in the mean tumour volume of D1) and

T0=treatment groups are in the mean tumour volume of D1.

The net tumour that negative T/T0 values represent a certain group is reduced.40% or smaller T/C values show potential therapeutic activity.

Tumor growth delay

Neoplasm reaches terminal volume (2000mm³) or reach research last day (D51) when, to each animal implement pacify It is happy dead.Reach each animal of terminal volume for tumour, terminal time (TTE) is calculated by following equation：

TTE=(log₁₀(terminal volume)-b)/m

Wherein TTE is represented with number of days, and terminal volume is with mm³It represents, b is intercept, and m is the tumour growth data of logarithmic transformation Collect the slope of line obtained by linear regression.Data set include be more than research terminal volume first observation and face reach terminal volume it Preceding continuous 3 observations.Any animal that tumour is not up to terminal is endowed the TTE values equal to research last day.It is classified as TTE values equal to dead day are endowed by the lethal any animal of TR reasons.It is classified as lethal (other than transfer) by NTR reasons Any animal excludes from TTE calculating.

Therapeutic efficiency is measured according to tumor growth delay (TGD), and TGD is defined as the intermediate value TTE that control group is compared by treatment group Increase：

TGD=T-C,

It is represented with number of days or the percentage of intermediate value TTE as a control group：

%TGD=[(T-C) C] x100

Wherein：

The intermediate value TTE of T=treatment groups,

C=specifies the intermediate value TTE of control group.

Subside the MTV reacted and standard

The animal tumor volume that therapeutic efficiency can be also remained according to last day in research and the reaction quantity that subsides To measure.MTV (n) is defined as the median tumor volume of D51 in the size of animal n retained, and the tumour of the animal is not up to eventually Point volume.Treatment can cause the partial remission (PR) of tumour in animal or subside (CR) completely.PR shows with regard to 3 times in flow of research For continuous measurement, gross tumor volume is 50% or less its D1 volume, and is equal to for one or more described 3 times measure Or more than 13.5mm³.CR shows that for 3 times in flow of research continuous measurements gross tumor volume is less than 13.5mm³.It is last in research Any animal that CR reactions are presented for one day is additionally classified as no tumour Survivor (TFS).

Toxicity

Animal is weighed day in the 1st -5 day, each treatment (in addition to weekend), 2 times a week later, until research terminates.Group is average BW minimum points measure can exclude that the measurement carried out after mouse is withdrawn from the study can be assessed more than 50% in group, also exclude to sort out in research Any animal for NTR death.The group that the acceptable toxicity of maximum tolerated dose (MTD) is defined as in experiment less than 15% is averaged BW loses, and it is dead to be no more than an example TR in 10 animals.BW losses are more than 15% or with regard to 1 survey for 3 continuous measurements Any animal of the BW losses more than 20% is subjected to euthanasia for amount, is classified as TR death, unless it is the first case in group It is dead.It is if damned not evidence show dead related to the treatment side effect and dead appearance more than 14 days after application It dies and is classified as non-treatment correlation (NTR).NTR death is classified as NTRa (being attributed to unexpected or mistake), NTRu (is attributed to unknown Reason) or NTRm (being spread by invading and/or shifting the tumour confirmed come postmortem).Maximum letter is provided simultaneously to preserve animal It ceases, the first case death in group is classified as NTRu；If had recorded in same group, 2 subsequent TR are dead, and the NTR death is again It is classified as TR.

Statistics and pattern analysis

All statistics and pattern analysis are carried out with the Prism 3.03 (GraphPad) for Windows.Group 1-7 is averaged Tumor volume change is compared first with variance analysis (ANOVA), uses bartlett's test.Have between bartlett's test prompting variance Significant difference (P<0.0001) when, which is compared with Ke-Wa Shi One-way ANOVAs, is had between display median tumor volume change aobvious Write difference (P<0.0001).Dunne's multiple comparative test later may compare medication therapy groups and control group 1.It is repeated once these Non-parametric test is to compare the corresponding single therapy of one of combination therapy group.

Survival is analyzed by Kapp Lan-Mei Ye methods.Logarithm order (Mantel-Cox) examines total life for analyzing two groups Deposit the conspicuousness of difference between experience (survivorship curve).The individual TTE of all animals in Log-Rank Test analysis group, in addition to those It is excluded as non-treatment related (NTR) death.Double tail statistical analyses are implemented under P=0.05.Prism is general by inspection result Include for：P>When 0.05 not significantly (ns), 0.01<Significantly (to be represented with symbol " * ") during P≤0.05,0.001<It is during P≤0.01 Very significantly (" * * "), for extremely significantly (" * * * ") during P≤0.001.Since statistical significance inspection does not provide group difference degree Estimation, the conspicuousness of all level are notable described in this report text or not notable.

As a result

According to above procedure, following results are obtained under study for action：

Study terminal=2000mm³, short-lived reactive=13 day, progress 51 in number of days.

Dead size of animal is not led to by accident or unknown cause in N=groups or implements the dynamic of euthanasia to sample Object quantity.

The mean tumour volume of T/C=100x (Δ T/ Δ C)=treatment group is compared % of the control group between D1 and D13 and is become Change.

T/T₀=100x (Δ T/T0)=and as Δ T=0, the mean tumour volume for the treatment of group compares its initial volume in D1 % variations between D13.

Statistical significance (Dunne's inspection)=P values from the unidirectional variance Dunne multiple comparative tests of Ke-Wa Shi：Ns=is not Significantly=P>0.05, compare shown group.

The shown group of P value from Log-Rank Test of statistical significance (Log-Rank Test)=compare：Ns=is not notable；P< Result is regarded as significantly when 0.05.

The c-Met inhibitor single therapies of 15mg/kg and 30mg/kg generate 5% and 1%T/C respectively.The change of 30mg/kg Close the T/C that object C generates 31%.T/T0 is realized in 15mg/kg c-Met inhibitor and the combination of 30mg/kg compounds C:- 83% Tumor regression, hence it is evident that better than any independent reagent.

After treatment terminates, tumor growth delay percentage is assessed by monitoring tumour growth and record terminal time (TTE) Than (%TGD).Supporting agent treatment group reached terminal at 11.7 days.The c-Met inhibitor of 15mg/kg and 30mg/kg makes intermediate value respectively TTE increases by 21.4 days (183%TGD) and 23.4 days (200%TGD).The compound C of 30mg/kg makes intermediate value TTE increase by 9.3 days (79%TGD).The combination of 15mg/kg c-Met inhibitor and 30mg/kg compounds C show the intermediate value TTE of 28.7 days, are 245%TGD.

Embodiment 6：The combination of compound D is used in non-small cell lung cancer heteroplastic transplantation model

In this experiment, NCI-H1993 Non-small cell lung carcinomas heteroplastic transplantation model is used to evaluate PI3K inhibitor compounds D With the fluoro- N- methyl -4- of c-Met inhibitor 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine -2 base] benzene Formamide is as single agents or the antitumor efficacy of combination.NCI- obtained from American Type Culture Collection (ATCC) H1993 tumour cells derive from patient's Metastatic Lymph Nodes, and the patient is with 3A phases adenocarcinoma of lung and has smoking history.

Mouse

In the D1 of research, female athymic nude mouse (Crl:NU(Ncr)-Foxn1^nu, Charles River) and for 8 week old, weight (BW) ranging from 16.1-26.2g.Animal can free water (reverse osmosis, 1ppm Cl), NIH 31Modified are freely eaten and Irradiated LabThe diet is made of 18.0% crude protein, 5.0% crude fat and 5.0% crude fibre. Mouse feeder is in irradiation Enrich-o ' cobs^TMOn the bedding and padding of laboratory, declined isolator using static state, in 20-22 DEG C (68- 72 °F) and 12 small time cycles of 40-60% humidity in.

Tumour transplatation and measurement

NCI-H1993 tumor cell lines, which are obtained from American Type Culture Collection and are maintained at, is dissolved in RPMI-1640 cultures The DRS_NC of base, the culture medium are big mould comprising 100 units/novocillin, 100 μ g/mL streptomycin sulfates, 25 μ g/mL celebratings Element, 10% fetal calf serum and 2mM glutamine.Tumour cell is cultivated in tissue culture flasks, uses 37 DEG C and atmosphere as 5% CO₂With the humidified incubator of 95% air.

It for the tumour cell of implementation and is resuspended in exponential phase harvest containing 50%Matrigel^TM(BD bioscience (BD Biosciences)) cold PBS.The right side oxter of each mouse is subcutaneously injected into 1x10⁷A cell (0.2mL cell suspensions). Tumour slide calliper rule are measured with 2 dimensions so as to monitoring growth, when its average external volume is close to required 200-250mm³During range.With mm³The tumor size of meter is calculated as below：Gross tumor volume=(width²X length)/2, wherein width and length is tumour (in terms of mm) Measurement.Tumor weight can be used it is assumed hereinafter that estimating：1mg is equal to 1mm³Gross tumor volume.14 days after tumor cell transplantation, in research D1, individual tumors volume are 172-256mm³Mouse be divided into every group of 10 mouse, group mean tumour volume is 206-215mm³。

Test specimen

The fluoro- N- methyl -4- of c-Met inhibitor 2- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine - 2 bases] benzamide (hydrochloride, 84.98% free alkali) 4 DEG C preserve and add in the deionized water equal to 1/2 final volume, ultrasound Wave processing is until dissolving obtains uniform 2X suspensions.Add in isometric 0.5% methylcellulose:0.1%80 with offer It is dissolved in the 1.77mg/mL solution of deionized water, a concentration of 0.25% methylcellulose of whole supporting agent and 0.05%80 (carry Agent 1).It is daily to prepare a fresh solution for p.m. applications and room temperature preservation, until a.m. is applied.

Compound D is suspended from the deionized water containing 0.5% methylcellulose with 3.0mg/mL.Branch point, which adds in, is equal to 1/3 eventually The supporting agent of volume.Suspension is vibrated after adding in every time, then final probe on ice to prepare uniform white hang by ultrasonication Liquid.Fresh suspension is prepared weekly, and 4 DEG C of preservations suspend again using preceding.

Prepare supporting agent 2, whole a concentration of 35%D5W of supporting agent (5% glucose for being dissolved in water):5%10%80 (0.05% final concentration):60%100mM acetate buffers pH 4.5.

Treatment plan

C-Met inhibitor and supporting agent are respectively by administration by gavage (p.o.) application twice daily until terminating during research.Change Object D is closed to apply once a day until terminating by administration by gavage during research.In combination therapy, compound D is in c-Met inhibitor It is applied in 30 minutes afterwards.In all groups, the applied volume (0.2mL/20g mouse) of 10mL/kg according to as apply surveyed day it is each Animal weight scales, the weekend in addition to continuing previous BW.

Nude mouse (n=10/ groups) is treated as follows.Group 1 receives supporting agent 1 and supporting agent 2, and as the control of research.Group 2 is being ground Receive 17.7mg/kg c-Met monotherapies twice daily during studying carefully (b.i.d. is until terminating).Group 3 receives during research 30mg/kg compound D monotherapies once a day (q.d. is until terminating).It is every that group 4 receives 17.7mg/kg c-Met inhibitor Day is twice until terminating with 30mg/kg compounds D once a day until the combination of end.

Tumor growth inhibition

Short term efficacy was measured at the 20th day (D20).Before D20,11 animal and 1 animal of group 3 are organized since tumour is sent out It opens up and exits；Respective end gross tumor volume continues to develop and is included in a group average external volume.

Measure tumor volume difference terminal of each animal in D1 (application starts) between terminal day.For each treatment group, The reaction of terminal day passes through the calculating of one of following relationship：

Wherein

Δ T=(treatment group is in the mean tumour volume of terminal day)-(treatment group is in the mean tumour volume of D1),

Δ C=(control group is in the mean tumour volume of terminal day)-(control group is in the mean tumour volume of D1) and

T0=treatment groups are in the mean tumour volume of D1.

The net tumour that negative T/T0 values represent a certain group is reduced.40% or smaller T/C values show potential therapeutic activity.

Tumor growth delay

Tumour is measured weekly secondary with slide calliper rule, and neoplasm reaches terminal volume (600mm³) or reach research last day (D71) when (whichever first occurs), to each euthanizing animals.The terminal time (TTE) of each mouse passes through following equation It calculates：

TTE=(log₁₀(terminal volume)-b)/m

Wherein TTE is represented with number of days, and terminal volume is represented with mm3, and b is intercept, and m is the tumour growth data of logarithmic transformation Collect the slope of line obtained by linear regression.Each data set includes being more than to study the first observation of terminal volume and face to reach terminal volume Continuous 3 observations before.Any animal that tumour is not up to terminal is endowed the TTE values equal to research last day.Sort out To be endowed the TTE values equal to dead day by the lethal any animal of related (TR) reason for the treatment of.It is classified as by non-treatment correlation (NTR) reason any animal lethal (other than transfer) excludes from TTE calculating.

Therapeutic efficiency is measured according to tumor growth delay (TGD), and TGD is defined as the intermediate value TTE that control group is compared by treatment group Increase：

TGD=T-C,

It is represented with number of days or the percentage of intermediate value TTE as a control group：

%TGD=[(T-C) C] x100

Wherein：

The intermediate value TTE of T=treatment groups,

C=specifies the intermediate value TTE of control group.

Subside the MTV reacted and standard

The animal tumor volume that therapeutic efficiency can be also remained according to last day in research and the reaction quantity that subsides To measure.MTV (n) is defined as the median tumor volume of D71 in the size of animal n retained, which is not up to terminal volume.

Treatment can cause the partial remission (PR) of tumour in animal or subside (CR) completely.PR shows with regard in flow of research 3 For secondary continuous measurement, gross tumor volume is 50% or less its D1 volume, and for one or more described 3 times measure etc. In or more than 13.5mm³.CR shows that for 3 times in flow of research continuous measurements gross tumor volume is less than 13.5mm³。

Toxicity

Animal is weighed day in the 1st -5 day, each treatment (in addition to weekend), 2 times a week later, until research terminates.With regard to D1 with The interval of D20 and complete research measure maximum group average weight (BW) and lose.The average BW minimum points measure of group can be excluded in group The measurement carried out after mouse is withdrawn from the study can be assessed more than 50%, also excludes to be classified as non-treatment related (NTR) death in research Any animal.The acceptable toxicity of maximum tolerated dose (MTD) be defined as experiment in less than 15% group be averaged BW lose, and It is dead to be no more than an example TR in 10 animals.More than 15% or for 1 time measures, BW is damaged for BW losses for 3 continuous measurements Any animal for quenching 20% is subjected to euthanasia, is classified as TR death, unless it is the first case death in group.If not yet Evidence show dead related to the treatment side effect and dead appearance more than 14 days after application, which is classified as NTR.NTR death is classified as NTRa (being attributed to unexpected or mistake), NTRu (being attributed to unknown cause) or NTRm and (passes through intrusion And/or transfer carrys out the tumour diffusion that postmortem confirms).Maximum information is provided simultaneously to preserve animal, and the first case in group is dead It is classified as NTRu；If had recorded in same group, 2 subsequent TR are dead, which reclassifies as TR.

Statistics and pattern analysis

All statistics and pattern analysis are carried out with the Prism 3.03 (GraphPad) for Windows.All groups flat Equal tumor volume change One-way ANOVA (ANOVA) compares, and bartlett's test is neat for variance.It is aobvious due to having between variance Write difference ((P<0.01), compared with Ke-Wa Shi One-way ANOVAs and the subsequent multiple comparative test of Dunne for described group.It repeats Inspection below is with the combination and the single therapy of its component.

Survival is analyzed by Kapp Lan-Mei Ye methods based on TTE values.Log-Rank Test passes through for analyzing two groups of total existence Go through the conspicuousness of difference between (survivorship curve).Double tail statistical analyses are implemented under P=0.05.Prism summarizes inspection result For：P>When 0.05 not significantly (ns), 0.01<Significantly (to be represented with symbol " * ") during P≤0.05,0.001<For very during P≤0.01 Significantly (" * * "), for extremely significantly (" * * * ") during P≤0.001.Since statistical significance inspection does not provide estimating for group difference degree Meter, the conspicuousness of all level are notable described in this report text or not notable.

As a result

According to above procedure, following results as defined above are obtained at the end of research：

Dead size of animal is not led to by treatment correlation, accident or unknown cause in N=groups or implements peace to sample Happy dead size of animal

The mean tumour volume of T/C=100x (Δ T/ Δ C)=treatment group is compared % of the control group between D1 and D20 and is become Change.

T/T₀During=100x (Δ T/T0)=Δ T=0, the mean tumour volume for the treatment of group compare its initial volume D1 with % variations between D13.

Statistical significance (Dunne's inspection)=P values from the unidirectional variance Dunne multiple comparative tests of Ke-Wa Shi：Ns=is not Significantly=P>0.05, compare shown group.

The shown group of P value from Log-Rank Test of statistical significance (Log-Rank Test)=compare：Ns=is not notable；P< Result is regarded as significantly when 0.05.

Since the overlapping of variable reactive and gross tumor volume range changes, with 17.7mg/kg gavages twice daily until terminating C-Met inhibitor single therapies significantly inhibiting compared to control (group 1) is not implemented.With 30mg/kg gavages once a day until The compound D single therapies of end cause inapparent median tumor Acceleration of growth.The combination of c-Met inhibitor and compound D Generation -17%T/T₀With notable intermediate value activity (P<0.01, Deng Nite examines), but it is single compared to c-Met inhibitor at the 20th day Treatment does not significantly improve.

Generally, c-Met inhibitor single therapy makes intermediate value TTE increase by 97% and significantly extends survival (P<0.001, it is right Number rank tests), generate 4 D71 survivors and 1 PR.Compared to control (group 1), well tolerable compound D single therapies generate The overall survival that unobvious shorten.

The combination of c-Met inhibitor and compound D make intermediate value TTE increase the 184% of maximum possible.The combination group has 8 Survivor and 1 partial remission.The combination is significantly improved compared to two kinds of single therapies.Fig. 5 of this paper provides the summary of result.

15%T/C and non-aobvious is generated (twice daily until terminating) in short, 17.7mg/kg c-Met inhibitor single therapy The D20 Tumor growth inhibitions of work, but intermediate value TTE is caused to increase by 97% and existence significantly extension.30mg/kg compounds D's is single Treatment (once a day until terminating) is without remarkable activity (inactivation).By causing in D20, tumour is reduced and generation maximum can 184% intermediate value TTE of energy increases, which shows advantageous drug interaction.It does not observe in D20 and inhibits compared to c-Met Agent single therapy significantly improves；However, the combination significantly extends survival relative to two kinds of corresponding single therapies.c-Met 2 death in 1 death and combination therapy in inhibitor single therapy are attributed to whose body weight loss.Due to organizing average body Whether loss is zero or can be neglected again, it is impossible to determine c-Met inhibitor single therapy in maximum whole dosage and the combination Whether more than the whole dosage of the maximum of model.

Claims (15)

1. a kind of pharmaceutical composition, including (a) (S)-pyrrolidines -1,2- dicarboxylic acids 2- amides 1- (4- methyl -5- [2- (2,2, Tri- fluoro- 1,1- dimethyl-ethyIs of 2-)-pyridin-4-yl]-thiazol-2-yl }-amide) i.e. compound D or its is pharmaceutically acceptable Salt and (b) at least one c-Met receptor tyrosine kinase inhibitors or its pharmaceutically-acceptable salts, for treating proliferative disease Disease.

2. pharmaceutical composition according to claim 1, it is characterised in that the c-Met receptor tyrosine kinase inhibitors are 2- fluoro- N- methyl -4- [7- quinoline -6- bases-methyl)-imidazo [1,2-b] [1,2,4] triazine -2 base] benzamide or its pharmaceutically Acceptable salt.

3. pharmaceutical composition according to claim 1, wherein the proliferative diseases are c-Met dependence proliferative diseases.

4. pharmaceutical composition according to claim 1, wherein the proliferative diseases are cancers.

5. pharmaceutical composition according to claim 1, wherein the proliferative diseases are benign and malignant tumours selected from the group below：Breast Gland, bladder, cervix, cholangiocarcinoma, Colon and rectum, oesophagus, stomach, head and neck, kidney, liver, lung, nasopharynx, ovary, pancreas, forefront Gland, thyroid gland, endometrium, musculoskeletal system sarcoma, soft tissue sarcoma, Huppert's disease, lymthoma, adult type T are thin Born of the same parents' leukaemia, acute myelogenous leukemia, chronic myelogenous leukemia, spongioblastoma, astrocytoma, melanoma, mesothelium Knurl and embryonal carcinosarcoma.

6. pharmaceutical composition according to claim 1, which is characterized in that the proliferative diseases are lung cancer or spongioblastoma.

7. pharmaceutical composition according to claim 6, which is characterized in that the lung cancer is non-small cell lung cancer.

8. according to the pharmaceutical composition described in any one of claim 1-7, which is characterized in that the composition is simultaneously, separately Or the combination of sequence application.

9. a kind of pharmaceutical composition, including the pharmaceutical composition described in claims 1 or 2 and at least one pharmaceutically acceptable load Body.

10. the pharmaceutical composition of claim 1, which is used to prepare, inhibits to suffer from the purposes of drug that the transfer in cancer object is formed, the suppression System includes applying the pharmaceutical composition as described in claim 1 of pharmaceutical effective amount to object in need.

11. purposes according to claim 10, wherein the cancer is c-Met dependence cancers.

12. according to the purposes of claim 10 or 11, which is characterized in that the proliferative diseases or cancer are selected from the group below good Property and malignant tumour：Mammary gland, bladder, cervix, cholangiocarcinoma, Colon and rectum, oesophagus, stomach, head and neck, kidney, liver, lung, nasopharynx, Ovary, pancreas, prostate, thyroid gland, endometrium, musculoskeletal system sarcoma, soft tissue sarcoma, Huppert's disease, leaching Bar knurl, adult T cell leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, spongioblastoma, astrocyte Knurl, melanoma, celiothelioma and embryonal carcinosarcoma.

13. according to the purposes of claim 10 or 11, wherein the therapeutic agent for inhibiting to include separating or sequentially apply the amount (a) and the therapeutic agent (b) of the amount.

14. a kind of pharmaceutical composition, the combination includes (S)-pyrrolidines -1,2- dicarboxylic acids 2- amides 1- ({ 4- methyl -5- [2- (tri- fluoro- 1,1- dimethyl-ethyIs of 2,2,2-)-pyridin-4-yl]-thiazol-2-yl-amide) i.e. compound D or its pharmaceutically may be used Receive salt and the fluoro- N- methyl -4- of c-Met receptor tyrosine kinase inhibitors 2- [7- quinoline -6- bases-methyl)-imidazo [1,2- B] -2 base of [1,2,4] triazine] benzamide or its pharmaceutically-acceptable salts.

15. pharmaceutical composition according to claim 14, for treating proliferative diseases, wherein the proliferative diseases are (a) c-Met dependences proliferative diseases or (b) cancer or (c) benign and malignant tumour selected from the group below：Mammary gland, bladder, uterus Neck, cholangiocarcinoma, Colon and rectum, oesophagus, stomach, head and neck, kidney, liver, lung, nasopharynx, ovary, pancreas, prostate, thyroid gland, son Endometrium, it is musculoskeletal system sarcoma, soft tissue sarcoma, Huppert's disease, lymthoma, adult T cell leukemia, acute Myelomatosis, chronic myelogenous leukemia, spongioblastoma, astrocytoma, melanoma, celiothelioma and embryonal carcinoma meat Knurl.